Bruton&#39;s tyrosine kinase inhibitor combinations and uses thereof

ABSTRACT

Disclosed herein are methods, compositions, and kits for treating a B-cell malignancy comprising administering a combination of a BTK inhibitor (e.g. ibrutinib) and a PIM inhibitor. Also disclosed herein are methods, compositions, and kits for treating a BTK-resistant B-cell malignancy comprising administering a combination of a BTK inhibitor (e.g. ibrutinib) and a PIM inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority to U.S. ProvisionalApplication No. 62/034,997, filed Aug. 8, 2014; U.S. ProvisionalApplication No. 62/082,972, filed Nov. 21, 2014; U.S. ProvisionalApplication No. 62/086,162, filed Dec. 1, 2014; and U.S. ProvisionalApplication No. 62/196,251, filed Jul. 23, 2015; all of whichapplications are incorporated herein by reference in their entireties.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING SUBMITTED AS A TEXT FILEVIA EFS-WEB

The instant application contains a Sequence Listing, which has beensubmitted as a computer readable text file in ASCII format via EFS-Weband is hereby incorporated in its entirety by reference herein. The textfile, created date of Aug. 6, 2015, is named 25922-315-201SEQ.TXT and is28,633 bytes in size.

BACKGROUND OF THE INVENTION

Bruton's tyrosine kinase (BTK), a member of the Tec family ofnon-receptor tyrosine kinases, is a key signaling enzyme expressed inall hematopoietic cells types except T lymphocytes and natural killercells. BTK plays an essential role in the B-cell signaling pathwaylinking cell surface B-cell receptor (BCR) stimulation to downstreamintracellular responses.

SUMMARY OF THE INVENTION

Disclosed herein, in certain embodiments, is a method of treating aB-cell malignancy in a subject in need thereof, that comprisesadministering to the subject a therapeutically effective amount of acombination comprising a BTK inhibitor and an anticancer agent, whereinthe anticancer agent inhibits MALT1, MCL-1, or IDH1. Also disclosedherein, in some embodiments, is a method of treating a BTKinhibitor-resistant B cell malignancy in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of a combination comprising a BTK inhibitor and an anticanceragent, wherein the anticancer agent inhibits MALT1, MCL-1, or IDH1. Insome embodiments, the combination provides a synergistic therapeuticeffect compared to administration of the BTK inhibitor or the anticanceragent alone. In some embodiments, the combination sensitizes a B-cellmalignancy to the BTK inhibitor. In some embodiments, the anticanceragent inhibits MALT1. In some embodiments, the anticancer agent thatinhibits MALT1 comprises MI-2, mepazine, thioridazine, and promazine. Insome embodiments, the anticancer agent inhibits MCL-1. In someembodiments, the anticancer agent that inhibits MCL-1 comprises BI97C10,BI112D1, gossypol, obatoclax, MG-132, MIM1, sabutoclax, and TW-37. Insome embodiments, the anticancer agent inhibits IDH1. In someembodiments, the anticancer agent that inhibits IDH1 comprises AGI-5198,AG-120, IDH-C227, and ML309. In some embodiments, the BTK inhibitor isibrutinib. In some embodiments, the B-cell malignancy is acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), acute monocytic leukemia (AMoL), chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risksmall lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuselarge B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom'smacroglobulinemia, multiple myeloma, extranodal marginal zone B celllymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma,non-Burkitt high grade B cell lymphoma, primary mediastinal B-celllymphoma (PMBL), immunoblastic large cell lymphoma, precursorB-lymphoblastic lymphoma, B cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cellmyeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma,intravascular large B cell lymphoma, primary effusion lymphoma, orlymphomatoid granulomatosis. In some embodiments, the B-cell malignancyis diffuse large B-cell lymphoma (DLBCL). In some embodiments, the DLBCLis activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL). In someembodiments, the B-cell malignancy is a relapsed or refractory B-cellmalignancy. In some embodiments, ibrutinib is administered once a day,two times per day, three times per day, four times per day, or fivetimes per day. In some embodiments, ibrutinib is administered at adosage of about 40 mg/day to about 1000 mg/day. In some embodiments,ibrutinib is administered orally. In some embodiments, ibrutinib and theanticancer agent are administered simultaneously, sequentially orintermittently. In some embodiments, the method further comprisesadministering a third therapeutic agent. In some embodiments, the thirdtherapeutic agent is selected from among a chemotherapeutic agent orradiation therapeutic agent. In some embodiments, the chemotherapeuticagent is selected from among chlorambucil, ifosfamide, doxorubicin,mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus,fludarabine, fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab,dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab,bortezomib, pentostatin, endostatin, or a combination thereof.

Disclosed herein, in certain embodiments, is a method of treating adiffuse large B-cell lymphoma (DLBCL) in a subject in need thereof, thatcomprises administering to the subject a therapeutically effectiveamount of a combination comprising a BTK inhibitor and an anticanceragent, wherein the anticancer agent inhibits MALT1, MCL-1, or IDH1. Insome embodiments, the combination provides a synergistic therapeuticeffect compared to administration of the BTK inhibitor or the anticanceragent alone. In some embodiments, the combination sensitizes a B-cellmalignancy to the BTK inhibitor. In some embodiments, the anticanceragent inhibits MALT1. In some embodiments, the anticancer agent thatinhibits MALT1 comprises MI-2, mepazine, thioridazine, and promazine. Insome embodiments, the anticancer agent inhibits MCL-1. In someembodiments, the anticancer agent that inhibits MCL-1 comprises BI97C10,BI112D1, gossypol, obatoclax, MG-132, MIM1, sabutoclax, and TW-37. Insome embodiments, the anticancer agent inhibits IDH1. In someembodiments, the anticancer agent that inhibits IDH1 comprises AGI-5198,AG-120, IDH-C227, and ML309. In some embodiments, the BTK inhibitor isibrutinib. In some embodiments, the DLBCL is activated B-cell diffuselarge B-cell lymphoma (ABC-DLBCL). In some embodiments, the DLBCL is arelapsed or refractory DLBCL. In some embodiments, ibrutinib isadministered once a day, two times per day, three times per day, fourtimes per day, or five times per day. In some embodiments, ibrutinib isadministered at a dosage of about 40 mg/day to about 1000 mg/day. Insome embodiments, ibrutinib is administered orally. In some embodiments,ibrutinib and the anticancer agent are administered simultaneously,sequentially or intermittently. In some embodiments, the method furthercomprises administering a third therapeutic agent. In some embodiments,the third therapeutic agent is selected from among a chemotherapeuticagent or radiation therapeutic agent. In some embodiments, thechemotherapeutic agent is selected from among chlorambucil, ifosfamide,doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus,everolimus, fludarabine, fostamatinib, paclitaxel, docetaxel,ofatumumab, rituximab, dexamethasone, prednisone, CAL-101, ibritumomab,tositumomab, bortezomib, pentostatin, endostatin, or a combinationthereof.

Disclosed herein, in certain embodiments, is a pharmaceuticalcombination that comprises (a) a BTK inhibitor; (b) an anticancer agent,wherein the anticancer agent inhibits MALT1, MCL-1 or IDH1; and (c) apharmaceutically-acceptable excipient. In some embodiments, thecombination provides a synergistic therapeutic effect compared toadministration of the BTK inhibitor or the anticancer agent alone. Insome embodiments, the combination sensitizes a B-cell malignancy to theBTK inhibitor. In some embodiments, the anticancer agent inhibits MALT1.In some embodiments, the anticancer agent that inhibits MALT1 comprisesMI-2, mepazine, thioridazine, and promazine. In some embodiments, theanticancer agent inhibits MCL-1. In some embodiments, the anticanceragent that inhibits MCL-1 comprises BI97C10, BI112D1, gossypol,obatoclax, MG-132, MIM1, sabutoclax, and TW-37. In some embodiments, theanticancer agent inhibits IDH1. In some embodiments, the anticanceragent that inhibits IDH1 comprises AGI-5198, AG-120, IDH-C227, andML309. In some embodiments, the BTK inhibitor is ibrutinib. In someembodiments, the combination is in a combined dosage form. In someembodiments, the combination is in separate dosage forms.

Disclosed herein, in certain embodiments, is a method of treating amantle cell lymphoma (MCL) in a subject in need thereof, that comprisesadministering to the subject a therapeutically effective amount of acombination comprising a BTK inhibitor and an anticancer agent, whereinthe anticancer agent is a MALT1 inhibitor or a proteasome inhibitor. Insome embodiments, the combination provides a synergistic therapeuticeffect compared to administration of the BTK inhibitor or the anticanceragent alone. In some embodiments, the combination sensitizes MCL to theBTK inhibitor. In some embodiments, the anticancer agent is a MALT1inhibitor. In some embodiments, the MALT1 inhibitor is selected fromMI-2, mepazine, thioridazine, or promazine. In some embodiments, theMALT1 inhibitor is MI-2. In some embodiments, the anticancer agent is aproteasome inhibitor. In some embodiments, the proteasome inhibitor isselected from carfilzomib or velcade. In some embodiments, MCL is arelapsed or refractory MCL. In some embodiments, MCL comprises amutation. In some embodiments, the mutation is a CARD11 mutation. Insome embodiments, the CARD11 mutation comprises a mutation at amino acidresidue position 225. In some embodiments, the mutation at amino acidresidue position 225 is a L225LI mutation. In some embodiments, theMALT1 inhibitor induces degradation of CARD11. In some embodiments, MCLis an ibrutinib-resistant MCL. In some embodiments, the BTK inhibitor isibrutinib. In some embodiments, ibrutinib is administered once a day,two times per day, three times per day, four times per day, or fivetimes per day. In some embodiments, ibrutinib is administered at adosage of about 40 mg/day to about 1000 mg/day. In some embodiments,ibrutinib is administered orally. In some embodiments, ibrutinib and theanticancer agent are administered simultaneously, sequentially orintermittently. In some embodiments, the method further comprisesadministering a third therapeutic agent. In some embodiments, the thirdtherapeutic agent is selected from among a chemotherapeutic agent orradiation therapeutic agent. In some embodiments, the chemotherapeuticagent is selected from among chlorambucil, ifosfamide, doxorubicin,mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus,fludarabine, fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab,dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab,bortezomib, pentostatin, endostatin, or a combination thereof.

Disclosed herein, in certain embodiments, is a method of treating anibrutinib-resistant mantle cell lymphoma (MCL) in a subject in needthereof, that comprises administering to the subject a therapeuticallyeffective amount of a combination comprising ibrutinib and an anticanceragent, wherein the anticancer agent is a MALT1 inhibitor or a proteasomeinhibitor. In some embodiments, the combination provides a synergistictherapeutic effect compared to administration of ibrutinib or theanticancer agent alone. In some embodiments, the combination sensitizesMCL to ibrutinib. In some embodiments, the anticancer agent is a MALT1inhibitor. In some embodiments, the MALT1 inhibitor is selected fromMI-2, mepazine, thioridazine, or promazine. In some embodiments, theMALT1 inhibitor is MI-2. In some embodiments, the anticancer agent is aproteasome inhibitor. In some embodiments, the proteasome inhibitor isselected from carfilzomib or velcade. In some embodiments, MCL comprisesa mutation. In some embodiments, the mutation is a CARD11 mutation. Insome embodiments, the CARD11 mutation comprises a mutation at amino acidresidue position 225. In some embodiments, the mutation at amino acidresidue position 225 is a L225LI mutation. In some embodiments, theMALT1 inhibitor induces degradation of CARD11. In some embodiments,ibrutinib is administered once a day, two times per day, three times perday, four times per day, or five times per day. In some embodiments,ibrutinib is administered at a dosage of about 40 mg/day to about 1000mg/day. In some embodiments, ibrutinib is administered orally. In someembodiments, ibrutinib and the anticancer agent are administeredsimultaneously, sequentially or intermittently. In some embodiments, themethod further comprises administering a third therapeutic agent. Insome embodiments, the third therapeutic agent is selected from among achemotherapeutic agent or radiation therapeutic agent. In someembodiments, the chemotherapeutic agent is selected from amongchlorambucil, ifosfamide, doxorubicin, mesalazine, thalidomide,lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib,paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone,CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin,or a combination thereof.

Disclosed herein, in certain embodiments, is a pharmaceuticalcombination that comprises (a) ibrutinib; (b) an anticancer agent,wherein the anticancer agent inhibits MALT1 or proteasome; and (c) apharmaceutically-acceptable excipient. In some embodiments, thecombination provides a synergistic therapeutic effect compared toadministration of ibrutinib or the anticancer agent alone. In someembodiments, the combination sensitizes MCL to ibrutinib. In someembodiments, the anticancer agent inhibits MALT1. In some embodiments,the anticancer agent that inhibits MALT1 comprises MI-2, mepazine,thioridazine, and promazine. In some embodiments, the anticancer agentthat inhibits MALT1 is MI-2. In some embodiments, the anticancer agentinhibits proteasome. In some embodiments, the anticancer agent thatinhibits proteasome comprises carfilzomib and velcade. In someembodiments, the combination is in a combined dosage form. In someembodiments, the combination is in separate dosage forms.

Disclosed herein, in certain embodiments, is a method of treating aB-cell malignancy in a subject in need thereof, comprising administeringto the subject a therapeutically effective amount of a combinationcomprising a BTK inhibitor and a PIM1 inhibitor. In some embodiments,the PIM1 inhibitor comprises mitoxantrone, SGI-1776, AZD1208, AZD1897,LGH447, JP_11646, Pim1 inhibitor 2, SKI-O-068, CX-6258, AR460770,AR00459339 (Array Biopharma Inc.), miR-33a, Pim-1 inhibitory p27 (Kip1)peptide, LY333′531, K00135, quercetagein (3,3′,4′,5,6,7-hydroxyflavone),or LY294002. In some embodiments, the B-cell malignancy is acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), acute monocytic leukemia (AMoL), chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risksmall lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuselarge B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom'smacroglobulinemia, multiple myeloma, extranodal marginal zone B celllymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma,non-Burkitt high grade B cell lymphoma, primary mediastinal B-celllymphoma (PMBL), immunoblastic large cell lymphoma, precursorB-lymphoblastic lymphoma, B cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cellmyeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma,intravascular large B cell lymphoma, primary effusion lymphoma, orlymphomatoid granulomatosis. In some embodiments, the B-cell malignancyis MCL. In some embodiments, MCL is primary-resistant MCL. In someembodiments, the BTK inhibitor is ibrutinib. In some embodiments,ibrutinib is administered once a day, two times per day, three times perday, four times per day, or five times per day. In some embodiments,ibrutinib is administered at a dosage of about 40 mg/day to about 1000mg/day. In some embodiments, ibrutinib is administered orally. In someembodiments, ibrutinib and the anticancer agent are administeredsimultaneously, sequentially or intermittently. In some embodiments, themethod further comprises administering a third therapeutic agent. Insome embodiments, the third therapeutic agent is selected from among achemotherapeutic agent or radiation therapeutic agent. In someembodiments, the chemotherapeutic agent is selected from amongchlorambucil, ifosfamide, doxorubicin, mesalazine, thalidomide,lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib,paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone,CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin,or a combination thereof.

Disclosed herein, in certain embodiments, is a method of treating mantlecell lymphoma (MCL) in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of acombination comprising a BTK inhibitor and a PIM1 inhibitor. In someembodiments, MCL is a primary-resistant MCL. In some embodiments, PIM1inhibitor comprises mitoxantrone, SGI-1776, AZD1208, AZD1897, LGH447,JP_11646, Pim1 inhibitor 2, SKI-O-068, CX-6258, AR460770, AR00459339(Array Biopharma Inc.), miR-33a, Pim-1 inhibitory p27 (Kip1) peptide,LY333′531, K00135, quercetagein (3,3′,4′,5,6,7-hydroxyflavone), orLY294002. In some embodiments, the BTK inhibitor is ibrutinib. In someembodiments, the method further comprises administering a thirdtherapeutic agent.

Disclosed herein, in certain embodiments, is a method of treating aB-cell malignancy in a subject in need thereof, comprising administeringto the subject a therapeutically effective amount of a combinationcomprising ibrutinib and a PIM1 inhibitor. In some embodiments, the PIM1inhibitor comprises mitoxantrone, SGI-1776, AZD1208, AZD1897, LGH447,JP_11646, Pim1 inhibitor 2, SKI-O-068, CX-6258, AR460770, AR00459339(Array Biopharma Inc.), miR-33a, Pim-1 inhibitory p27 (Kip1) peptide,LY333′531, K00135, quercetagein (3,3′,4′,5,6,7-hydroxyflavone), orLY294002. In some embodiments, the B-cell malignancy is acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), acute monocytic leukemia (AMoL), chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risksmall lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuselarge B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom'smacroglobulinemia, multiple myeloma, extranodal marginal zone B celllymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma,non-Burkitt high grade B cell lymphoma, primary mediastinal B-celllymphoma (PMBL), immunoblastic large cell lymphoma, precursorB-lymphoblastic lymphoma, B cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cellmyeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma,intravascular large B cell lymphoma, primary effusion lymphoma, orlymphomatoid granulomatosis. In some embodiments, the B-cell malignancyis MCL. In some embodiments, MCL is primary-resistant MCL. In someembodiments, the method further comprises administering a thirdtherapeutic agent.

Disclosed herein, in certain embodiments, is a method of treating mantlecell lymphoma (MCL) in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of acombination comprising ibrutinib and a PIM1 inhibitor. In someembodiments, MCL is primary-resistant MCL. In some embodiments, the PIM1inhibitor comprises mitoxantrone, SGI-1776, AZD1208, AZD1897, LGH447, JP11646, Pim1 inhibitor 2, SKI-O-068, CX-6258, AR460770, AR00459339 (ArrayBiopharma Inc.), miR-33a, Pim-1 inhibitory p27 (Kip1) peptide,LY333′531, K00135, quercetagein (3,3′,4′,5,6,7-hydroxyflavone), orLY294002. In some embodiments, the method further comprisesadministering a third therapeutic agent.

Disclosed herein, in certain embodiments, is a pharmaceuticalcombination comprising: (a) a BTK inhibitor; (b) a PIM1 inhibitor; and(c) a pharmaceutically-acceptable excipient. In some embodiments, thecombination provides a synergistic therapeutic effect compared toadministration of ibrutinib or the PIM1 inhibitor alone. In someembodiments, the combination sensitizes a hematological malignancy tothe BTK inhibitor. In some embodiments, the PIM1 inhibitor comprisesmitoxantrone, SGI-1776, AZD1208, AZD1897, LGH447, JP_11646, Pim1inhibitor 2, SKI-O-068, CX-6258, AR460770, AR00459339 (Array BiopharmaInc.), miR-33a, Pim-1 inhibitory p27 (Kip1) peptide, LY333′531, K00135,quercetagein (3,3′,4′,5,6,7-hydroxyflavone), or LY294002. In someembodiments, the BTK inhibitor is ibrutinib. In some embodiments, thecombination is in a combined dosage form. In some embodiments, thecombination is in separate dosage forms.

Disclosed herein, in certain embodiments, is a pharmaceuticalcombination comprising: (a) ibrutinib; (b) a PIM1 inhibitor; and (c) apharmaceutically-acceptable excipient. In some embodiments, the PIM1inhibitor comprises mitoxantrone, SGI-1776, AZD1208, AZD1897, LGH447,JP_11646, Pim1 inhibitor 2, SKI-O-068, CX-6258, AR460770, AR00459339(Array Biopharma Inc.), miR-33a, Pim-1 inhibitory p27 (Kip1) peptide,LY333′531, K00135, quercetagein (3,3′,4′,5,6,7-hydroxyflavone), orLY294002. In some embodiments, the combination is in a combined dosageform. In some embodiments, the combination is in separate dosage forms.

In some embodiments, a method of treating a hematological malignancy ina subject in need thereof is provided. The method includes the step ofadministering a therapeutically effective amount of a combinationcomprising a BTK inhibitor and a PIM inhibitor. Preferably, thecombination provides a synergistic effect compared to administration ofthe BTK inhibitor or the PIM inhibitor alone. Preferably, thecombination sensitizes the B-cell malignancy to the BTK inhibitor. Insome embodiments, the BTK inhibitor is ibrutinib. In some embodiments,the PIM inhibitor comprises mitoxantrone, SGI-1776, AZD1208, AZD1897,LGH447, JP_11646, Pim1 inhibitor 2, SKI-O-068, CX-6258, AR460770,AR00459339 (Array Biopharma Inc.), miR-33a, Pim-1 inhibitory p27 (Kip1)peptide, LY333′531, K00135, quercetagein (3,3′,4′,5,6,7-hydroxyflavone),or LY294002. In some embodiments, the PIM inhibitor is AZD1208. In someembodiments, the PIM inhibitor is a PIM1 inhibitor. In some embodiments,the PIM inhibitor is a pan-PIM inhibitor. In some embodiments, thehematological malignancy is a B-cell malignancy. In some embodiments,the B-cell malignancy is acute lymphoblastic leukemia (ALL), acutemyelogenous leukemia (AML), chronic myelogenous leukemia (CML), acutemonocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma (SLL), high-risk small lymphocytic lymphoma (SLL),follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantlecell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma,extranodal marginal zone B cell lymphoma, nodal marginal zone B celllymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma,primary mediastinal B-cell lymphoma (PMBL), immunoblastic large celllymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocyticleukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma,plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B celllymphoma, intravascular large B cell lymphoma, primary effusionlymphoma, or lymphomatoid granulomatosis. In some embodiments, the DLBCLis activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL). In someembodiments, the DLBCL is germinal center B-cell like DLBCL. In someembodiments, the B-cell malignancy is relapsed or refractory B-cellmalignancy. The PIM inhibitor may be administered simultaneously,sequentially, or intermittently. In some embodiments, the method furthercomprises administering a third therapeutic agent.

In some embodiments, a method of treating a diffuse large B-celllymphoma (DLBCL) in a subject in need thereof, comprising administeringto the subject a therapeutically effective amount of a combinationcomprising a BTK inhibitor and a PIM inhibitor. In some embodiments, thePIM inhibitor is a PIM1 inhibitor. In some embodiments, the PIMinhibitor is a pan-PIM inhibitor.

In some embodiments, a method of treating a BTK inhibitor-resistantB-cell malignancy in a subject in need thereof, comprising administeringto the subject a therapeutically effective amount of a combinationcomprising a BTK inhibitor and a PIM inhibitor. In some embodiments, thePIM inhibitor is a PIM1 inhibitor. In some embodiments, the PIMinhibitor is a pan-PIM inhibitor. In some embodiments, the combinationprovides a synergistic effect compared to administration of the BTKinhibitor or the PIM inhibitor alone. In some embodiments, thecombination sensitizes the BTK inhibitor-resistant B-cell malignancy tothe BTK inhibitor. In some embodiments, the BTK inhibitor is ibrutinib.

In some embodiments, a pharmaceutical composition is provided. Thepharmaceutical composition may include (a) a BTK inhibitor; (b) a PIMinhibitor; and (c) a pharmaceutically-acceptable excipient. In someembodiments, the PIM inhibitor is a PIM1 inhibitor. In some embodiments,the PIM inhibitor is a pan-PIM inhibitor. In some embodiments, the PIMinhibitor comprises mitoxantrone, SGI-1776, AZD1208, AZD1897, LGH447,JP_11646, Pim1 inhibitor 2, SKI-O-068, CX-6258, AR460770, AR00459339(Array Biopharma Inc.), miR-33a, Pim-1 inhibitory p27 (Kip1) peptide,LY333′531, K00135, quercetagein (3,3′,4′,5,6,7-hydroxyflavone), orLY294002. In some embodiments, the PIM inhibitor is AZD1208. In someembodiments, the PIM inhibitor is AZD1208.

In some embodiments, a method of selecting an individual having a B-cellmalignancy for therapy with a combination comprising a BTK inhibitor anda PIM inhibitor is provided. The method includes the steps of (a)measuring an expression level of PIM1 in a sample from the individual;(2) comparing the expression level of PIM1 with a reference level; and(3) characterizing the individual as a candidate for therapy with thecombination comprising a BTK inhibitor and a PIM inhibitor if theindividual has an elevated level of PIM1 compared to the referencelevel. In some embodiments, the elevated level of PIM1 is -fold,1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold,45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold,85-fold, 90-fold, 95-fold, 100-fold, or higher compared to theexpression of the reference level. In some embodiments, the referencelevel is the expression level of PIM1 in an individual who does not havea B-cell malignancy.

In some embodiments, a method of assessing whether a subject having aB-cell malignancy is less responsive or likely to become less responsiveto therapy with a BTK inhibitor. The method includes the steps of (a)testing a sample containing a nucleic acid molecule encoding a PIM1polypeptide from the subject; determining whether the encoded PIM1polypeptide is modified at an amino acid position 2, 81, or 97 of theamino acid sequence set forth in SEQ ID NO:1; and (c) characterizing thesubject as resistant or likely to become resistant to therapy with a BTKinhibitor if the subject has the modification at amino acid position 2,81, or 97. In some embodiments, the modification comprises asubstitution, an addition, or a deletion of the amino acid at amino acidposition 2, 81, or 97 in the PIM1 polypeptide. In some embodiments, themodification in the PIM1 polypeptide is selected from among PIM L2V,PIM1 P821S, or PIM1 S97N.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the invention are set forth with particularity in theappended claims. A better understanding of the features and advantagesof the present invention will be obtained by reference to the followingdetailed description that sets forth illustrative embodiments, in whichthe principles of the invention are utilized, and the accompanyingdrawings of which:

FIG. 1A and FIG. 1B illustrate the interaction properties of ibrutinibin combination with MCL-1, MALT1, IDH1, and JAK3 inhibitors.

FIG. 2A and FIG. 2B illustrate CARD11 mutation observed inibrutinib-resistant MCL patients.

FIG. 3 illustrates wild-type or mutant CARD11 expression in thepresences or absence of ibrutinib in Jeko cells. Mut2 is L224P mutation.Mut10 is L225LI mutation.

FIG. 4A and FIG. 4B illustrate the percentage of proliferation of Jekocells containing wild-type or mutant CARD11. Mut2 is L224P mutation.Mut10 is L225LI mutation.

FIG. 5A-FIG. 5C illustrate endogenous, over-expressed, and total levelsof CARD11 in Jeko cells by real-time PCR.

FIGS. 6A-D illustrate the percentage of inhibition of Jeko cells in thepresence of ibrutinib alone (FIG. 6A) or a combination of ibrutinib andvelcade (FIG. 6B), MI2 (FIG. 6C), or carlfilzomib (FIG. 6D).

FIG. 7A and FIG. 7B illustrate the percentage of proliferation ofOCI-Ly3 cells in the presence of a combination of ibrutinib with eithercarfilzomib or MI2.

FIG. 8 illustrates inhibition of BCR signaling by the combination ofibrutinib and MI2 in Jeko cells containing a L225LI (mut10) CARD11mutation.

FIG. 9 illustrates patient breakdown from the clinical trial MCL2001.Patients were further classified as progressive disease, moderateclinical benefit, and responders.

FIG. 10 illustrates patient breakdown based on clinical characteristics.

FIG. 11 illustrates genes associated with primary resistant, moderatebenefit, and responders.

FIG. 12 illustrates analysis of genes in primary nonresponders.

FIG. 13 illustrates a classification scheme of genes described herein.

FIG. 14A and FIG. 14B illustrate a graphical representation of PIM1pathway (FIG. 14A) and overall survival analysis from date of diagnosiscomprising either PIM1 expression (PIM pos) or no PIM1 expression (PIMneg) (FIG. 14B).

FIG. 15 illustrates schematics of NF-κB pathways that are modulated bymutations described herein.

FIG. 16A-FIG. 16C illustrate the endogenous, relative expression of PIM1(FIG. 16A), PIM2 (FIG. 16B), and PIM3 (FIG. 16C) genes in various celllines. (The y-axis is the relative gene expression). HBL1, TMD8,OCI-LY3, OCI-LY10, SU-DHL-2, and U-2932 are ABC-DLBCL cell lines.OCI-LY8, OCI-LY19, RCK-8, SU-DHL-1, SU-DHL-4, SU-DHL-5, SU-DHL-6,SU-DHL-8, SU-DHL-10, WSU-NHL, DB, HT, RL, and Toledo are GCB-DLBCL celllines.

FIG. 17 illustrates the relative cell growth of TMD8 and TMD8-colonycells when each are treated with ibrutinib.

FIG. 18 illustrates the relative gene expression of various genes,including PIM1. The relative gene expression depicted by the bar graphis a ratio of the gene expression in TMD8-colony cells vs. the geneexpression in TMD8 cells.

FIG. 19A illustrates the relative gene expression of PIM1, PIM2, andPIM2 in TMD8-WT and TMD8-ibrutinib-resistant cells (depicted as“TMD-resistant” in the graph). FIG. 19B illustrates the proteinexpression of PIM1 in TMD8-WT and TMD8-resistant, as well as HBL1-WT andHBL1-ibrutinib-resistant, cells. The “R” in figure refers to“resistant.”

FIG. 20A shows the synergy score of the drug dose matrix data for a cellviability assay in HBL1-WT cells grown in the presence of PIM inhibitor(AZD1208), ibrutinib, or a combination of the two drugs. The numbers inthe plot indicate a percentage of growth inhibition of cells treated for3 days with the corresponding compound combination relative to vehiclecontrol-treated cells. FIG. 20B shows the corresponding isobologram, inwhich points to the left of the diagonal line represent synergisticcombinations.

FIG. 21A shows the shows the synergy score of the drug dose matrix datafor a cell viability assay in HBL-1-ibrutinib-resistant(“HBL1-resistant”) cells grown in the presence of PIM inhibitor(AZD1208), ibrutinib, or a combination of the two drugs. The numbers inthe plot indicate a percentage of growth inhibition of cells treated for3 days with the corresponding compound combination relative to vehiclecontrol-treated cells. FIG. 21B shows the corresponding isobologram, inwhich points to the left of the diagonal line represent synergisticcombinations.

FIG. 22A shows that the combination of PIM inhibitor, AZD1208, andibrutinib enhanced the growth suppression effect of ibrutinib on HBL1-WTcells. FIG. 22B shows that the combination of PIM inhibitor, AZD1208,and ibrutinib enhanced growth suppression in HBL1-resistant(ibrutinib-resistant) cells compared to ibrutinib alone. FIG. 22Cillustrates the synergy score of the PIM inhibitor (AZD1208) andibrutinib combination in HBL1-WT and HBL1-resistant cells.

FIG. 23 shows that the combination of PIM inhibitor, AZD1208, andibrutinib enhanced the colony-reduction effect of ibrutinib in HBL1-WTcells compared to ibrutinib alone. At each concentration of ibrutinib,the following concentrations of PIM inhibitor were used: 0 nM (leftbar); 100 nM (middle bar); and 1000 nM (right bar).

FIGS. 24A-FIG. 24E show that the combination of the PIM inhibitor,AZD1208, and ibrutinib, enhanced the growth suppression effect ofibrutinib in HBL1 tumors. Shown are plots of tumor size over time forindividual animals treated with vehicle (FIG. 24B), ibrutinib (FIG.24C); PIM inhibitor (FIG. 24D); or a combination of ibrutinib and PIMinhibitor (FIG. 24E).

FIG. 25A is a chart showing various mutains in the PIM1 polypeptidefound in 6 DLBCL patients. The clinical response of each patient toibrutinib is indicated in the chart. FIG. 25B is a schematic showing thekinase domain of the PIM1 polypeptide, as well as a list of mutationsfound in the PIM1 polypeptide amongst DLBCL patients.

FIG. 26 is a schematic of a plasmid vector (construct) that can be usedto infect a cell line, such as a 293T cell line. PIM1 WT or mutant PIM1genes (i.e., PIM1 L2V, PIM1 P81S, and PIM1 297N) can be inserted intothe multiple cloning site (MCS) within the construct and stably infectedinto a cell line.

FIG. 27A-FIG. 27E show the results of a cycloheximide assay. 293T cellswere transduced with constructs having genes encoding PIM1-WT or PIM1L2V, PIM1 P81S, and PIM1 297N. The results indicate that PIM1 L2V; PIM1P81S; and PIM1 S97N are more stable than PIM1-WT proteins. FIG. 27E is agraph of the relative PIM1 protein expression (%) (y-axis) as a functionof time.

FIG. 28 is a graphical representation of the relative cell growth ofTMD8 cells transduced with constructs having genes encoding PIM-WT, PIM1L2V, PIM1 P81S, or PIMS97N, and treated with ibrutinib. As shown in thegraphs, TMD8 cells transduced with genes encoding PIM1 L2V, PIM1 P81S,or PIMS97N, were more resistant to ibrutinib.

FIGS. 29A-B show that PIM1-WT- and PIM1-mutant-transduced cells havesimilar cell growth and viability. FIG. 29A is a graphicalrepresentation of the cell growth of TMD8 cells transduced withcontructs having genes encoding PIM1-WT; PIM1 L2V; PIM1 P81S; or PIMS97N(with no drug treatment). FIG. 29B is a graphical representation of cellviability of TMD8 cells transduced with contructs having genes encodingPIM1-WT; PIM1 L2V; PIM1 P81S; or PIMS97N (with no drug treatment).

FIGS. 30A-30F show the results of a clonogenic cell survival assayperformed to evaluate whether any differences in the ability toproliferate indefinitely exists amongst the different modified TMD8 celllines.

DETAILED DESCRIPTION OF THE INVENTION

Methods, compositions, kits, and reagents are provided herein for use intreating a B-cell malignancy in a subject comprising administering tothe subject a therapeutically effective amount of a combinationcomprising a BTK inhibitor and an anticancer agent. Also disclosedherein, in some embodiments, are methods of treating a BTKinhibitor-resistant B cell malignancy in a subject, comprisingadministering to the subject a therapeutically effective amount of acombination comprising a BTK inhibitor and an anticancer agent. Furtherdisclosed herein, in some embodiments, are methods of treating a diffuselarge B-cell lymphoma (DLBCL) in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of acombination comprising a BTK inhibitor and an anticancer agent. In somecases, the anticancer agent inhibits MALT1, MCL-1, or IDH1. In somecases, the BTK inhibitor is ibrutinib.

Disclosed herein, in some embodiments, are pharmaceutical combinationscomprising a BTK inhibitor, an anticancer agent, and apharmaceutically-acceptable excipient. In some embodiments, theanticancer agent inhibits MALT1, MCL-1 or IDH1.

Certain Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. It is to be understoodthat the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictiveof any subject matter claimed. In this application, the use of thesingular includes the plural unless specifically stated otherwise. Itmust be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. In this application, theuse of “or” means “and/or” unless stated otherwise. Furthermore, use ofthe term “including” as well as other forms, such as “include”,“includes,” and “included,” is not limiting.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 μL” means “about 5 μL” and also “5 μL.” Generally, the term“about” includes an amount that would be expected to be withinexperimental error.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

“Antibodies” and “immunoglobulins” (Igs) are glycoproteins having thesame structural characteristics. The terms are used synonymously. Insome instances the antigen specificity of the immunoglobulin may beknown.

The term “antibody” is used in the broadest sense and covers fullyassembled antibodies, antibody fragments that can bind antigen (e.g.,Fab, F(ab′)2, Fv, single chain antibodies, diabodies, antibody chimeras,hybrid antibodies, bispecific antibodies, humanized antibodies, and thelike), and recombinant peptides comprising the forgoing.

The terms “monoclonal antibody” and “mAb” as used herein refer to anantibody obtained from a substantially homogeneous population ofantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts.

Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light and heavy-chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies.Variable regions confer antigen-binding specificity. However, thevariability is not evenly distributed throughout the variable domains ofantibodies. It is concentrated in three segments called complementaritydetermining regions (CDRs) or hypervariable regions, both in the lightchain and the heavy-chain variable domains. The more highly conservedportions of variable domains are celled in the framework (FR) regions.The variable domains of native heavy and light chains each comprise fourFR regions, largely adopting a β-pleated-sheet configuration, connectedby three CDRs, which form loops connecting, and in some cases formingpart of, the β-pleated-sheet structure. The CDRs in each chain are heldtogether in close proximity by the FR regions and, with the CDRs fromthe other chain, contribute to the formation of the antigen-binding siteof antibodies (see, Kabat et al. (1991) NIH PubL. No. 91-3242, Vol. I,pages 647-669). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as Fc receptor (FcR) binding, participation of theantibody in antibody-dependent cellular toxicity, initiation ofcomplement dependent cytotoxicity, and mast cell degranulation.

The term “hypervariable region,” when used herein, refers to the aminoacid residues of an antibody that are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarily determining region” or “CDR” (i.e., residues 24-34(L1), 50-56 (L2), and 89-97 (L3) in the light-chain variable domain and31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy-chain variabledomain; Kabat et al. (1991) Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institute of Health,Bethesda, Md.) and/or those residues from a “hypervariable loop” (i.e.,residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light-chainvariable domain and (H1), 53-55 (H2), and 96-101 (13) in the heavy chainvariable domain; Clothia and Lesk, (1987) J. Mol. Biol., 196:901-917).“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues, as herein deemed.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen-binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab, F(ab′)2, andF_(V) fragments; diabodies; linear antibodies (Zapata et al. (1995)Protein Eng. 10:1057-1062); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (C_(H1)) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain C_(H1) domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. Fab′ fragments are produced by reducing theF(ab′)2 fragment's heavy chain disulfide bridge. Other chemicalcouplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of human immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.Different isotypes have different effector functions. For example, humanIgG1 and IgG3 isotypes have ADCC (antibody dependent cell-mediatedcytotoxicity) activity.

“Anticancer agent” as used herein can refer to an inhibitor of MCL-1,MALT1, IDH1, or JAK3. “Anticancer agent” can also refer to a PIMinhibitor.

Overview

Hematological malignancy is a diverse group of cancer that affects theblood, bone marrow, and lymph nodes. It arises from an accumulation ofgenetic and epigenetic aberrations. For example, cancers of thehematopoietic cells develop resistance to growth-inhibitory anddifferentiation factors, proliferate in the absence of exogenous growthsignals, inhibit apoptosis, and evade immunosurveillance. Further,mutations within proteins that regulate these cellular functions areoften observed and these mutational disruptions involve proteins inpathways such as, for example, the BCR pathway, the NF-_(K)B pathway,and the JAK/STAT pathway, as well as proteins that regulate epigeneticalterations.

B-cell receptor (BCR) complex and its associated proteins play animportant role in the development, proliferation and survival of normalor malignant B cells. BCR function is required for normal antibodyproduction and abnormal BCR signal transduction is implicated in B-cellmalignancies. BCR signal transduction operates through several signalingpathways, including the PLCγ/calcium/NFAT pathway, the PI3K pathway, theIKK/NF-_(K)B pathway and the canonical ERK pathway. In some cases,chronic active B-cell receptor (BCR) signaling leads to constitutiveNF-_(K)B signaling, which in some cases, further leads to inhibition ofapoptosis.

The NF-_(K)B contributes to regulation of genes that control cellproliferation and cell survival. Under normal condition in unstimulatedcells, NF-_(K)B is sequestered in the cytoplasm by the I_(K)Bαinhibitor, which inactivates NF-_(K)B by masking the nuclearlocalization signals on NF-_(K)B. Upon stimulation, I_(K)Bα is degraded,which frees NF-_(K)B to enter into the nucleus, and subsequentlyupregulate genes that favor cell cycle progression, survival, cytokinesecretion, and inflammation. In cancerous cells, NF-_(K)B and theNF-_(K)B pathway are affected by oncogenic mutations, translocations,and copy number alterations that lead to constitutive signaling of theNF-_(K)B pathway.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1(MALT1) forms a complex with caspase recruitment domain family, member11 (CARD11 or CARMA1) and BCL10 (known as the CBM complex) to serve as asignaling scaffold that recruits TRAF6, TAK1, and the IKK complex toactivate the I_(K)B kinase β and thereby stimulates the NF-_(K)B throughthe classical pathway. In addition, MALT1 contains a paracaspase domainthat cleaves and inactivates negative regulators of canonical NF-_(K)Bsuch as A20, CYLD, and the NF-_(K)B subunit RelB which counteractpro-survival functions. Inhibition of protease activity of MALT1 as wellas mutation of the catalytic cysteine residue at position 464 lead toimpaired NF-_(K)B activation (Duwel, et al. “A20 negatively regulates Tcell receptor signaling to NF-kappaB by cleaving Malt1 ubiquitinchains,” J. Immunol. 182:7718-7728 (2009)). Further, the CBM complex hasbeen observed to be critical in regulating NF-_(K)B activation in cancersuch as Hodgkin lymphoma, multiple myeloma, marginal zone lymphoma, anddiffuse large B-cell lymphoma (DLBCL). Indeed, inhibition of the MALT1proteolytic activity by Z-VRPR-FMK, a polypeptide inhibitor, inhibitsNF-_(K)B dependent gene expression and exerts toxic effects in ABC-DLBCLcells (Ferch, et al., “Inhibiton of MALT1 protease activity isselectively toxic for activated B cell-like diffuse large B celllymphoma cells,” J. Exp. Med. 206:2313-2320 (2009); Hailfinger, et al.,“Essential role of MALT1 protease activity in activated B cell-likediffuse large B-cell lymphoma,” PNAS 106:19946-19951 (2009)).

The intrinsic apoptotic pathway is tightly regulated by members of theBcl-2 family. Several protein members share a homologous BH3 domain, andare referred to as the BH3-only proteins. These BH3-only proteins (e.g.BID, BAD, BIM, PUMA, and NOXA) are activated by cellular stress anddeath signals and promote the activation of oligomerization of thepro-apoptotic effectors BAX and BAK. BAX and BAK oligomerization leadsto mitochondrial outer membrane permeabilization, an event thatfacilitates a plethora of downstream activities leading to caspaseactivation and cellular destruction. Additional members of the Bcl-2family include pro-apoptotic proteins (e.g. BAX, BAK, and BOK) thatshare the BH domain, and anti-apoptotic proteins.

Myeloid cell leukemia 1 (MCL-1) is a member of the anti-apoptoticsubgroup of Bcl-2. MCL-1's expression and degradation is tightlyregulated in response to a variety of growth factors and glucosesignaling cascades, which might contributed to its short half-life,about 2-4 hours in most cells. Further, MCL-1 contributes to thesurvival of multiple cell lineages including lymphocytes (Opferman, etal. “Development and maintenance of B and T lymphocytes requiresantiapoptotic MCL-1,” Nature 426(6967):671-676 (2003); Dzhagalov, et al,“The anti-apoptotic Bcl-2 family member Mcl-1 promotes T lymphocytesurvival at multiple stages,” J. Immunol. 181(1):521-528 (2008)) andhematopoietic stem cells (Opferman, et al. “Obligate role ofanti-apoptotic MCL-1 in the survival of hematopoietic stem cells,”Science 307(5712):1101-1104 (2005)).

Under normal conditions, the anti-apoptotic MCL-1 sequesters Bak, anapoptotic effector protein, and the pro-apoptotic member Bim, therebypreventing cell death. However, upon cellular damage due to externalstimuli (e.g. UV irradiation or chemical agents), MCL-1 is untetheredfrom Bak and Bim, leading to cell death. During this process, both theMCL-1 gene is downregulated at a transcription level and the MCL-1protein degradation is enhanced. Under abnormal conditions, MCL-1 isupregulated, and in some cases, its overexpression has been associatedto chemotherapeutic resistance and relapse. In some instances, MCL-1might be critical in the survival of several types of malignancies. Forexample, MCL-1 is critical for the development and maintenance of acutemyeloid leukemia (Glaser et al., “Anti-apoptotic Mcl-1 is essential forthe development and sustained growth of acute myeloid leukemia,” GenesDev. 26(2):120-125 (2012); Xiang, et al. “Mcl1 hapoinsufficiencyprotects mice from Myc-induced acute myeloid leukemia,” J. Clin. Invest.120(6):2109-2118 (2010)). In addition, MCL-1 overexpression acceleratesMyc-induced lymphomagenesis (Campbell, et al., “Elevated Mcl-1 perturbslymphopoiesis, promotes transformation of hematopoietic stem/progenitorcells, and enhances drug resistance,” Blood 116(17):3197-3207 (2010)).Genetic ablation of MCL-1 gene has been shown to induce cell death inseveral types of cancer cells regardless of the expression of otheranti-apoptotic family members, such as in acute myeloid leukemia (Glaseret al., “Anti-apoptotic Mcl-1 is essential for the development andsustained growth of acute myeloid leukemia,” Genes Dev. 26(2):120-125(2012); Xiang, et al. “Mcl1 hapoinsufficiency protects mice fromMyc-induced acute myeloid leukemia,” J. Clin. Invest. 120(6):2109-2118(2010)).

Isocitrate dehydrogenase I (IDH1) belong to the family of isocitratedehydrogenases that catalyze the oxidative decarboxylation of isocitrateto α-ketoglutarate (α-KG), and the reduction of NADP⁺ to NADPH. Twoadditional members are present, IDH2, which shares a sequence andstructural similarity with IDH1, and IDH3, which participates inregulation of the TCA cycle. IDH1 is localized in the cytoplasm and theperoxisome. Mutations within IDH1 cause the reduction of α-KG toD-2-hydroxyglutarate (2-HG), which acts as an oncometabolite through theinhibition of α-KG-dependent enzymes, and stimulation of angiogenesis.Further, increased level of 2-HG leads to inhibition of α-KG-dependentenzyme ten-eleven-translocation 2 (TET2), which is responsible forcatalyzing the conversion of 5-methylcytosine (5mC) to5-hydrozymethylcytosine (5 hmC). Global accumulation of 5mC causesderegulation of the expression of genes through aberration of their CpGisland methylation, genome wide histone modifications, and DNA damagewith hypermethylation.

Mutation in IDH1 generally centered on the residue 132. In general,arginine at 132 has been substituted to histidine, serine, cysteine,glycine, or leucine. In addition, R132 mutation has been observed indifferent cancer types such as acute myeloid leukemia (AML), and acutelymphoid leukemia (ALL) (Paschka, et al., “IDH1 and IDH2 mutations arefrequent genetic alterations in acute myeloid leukemia and conferadverse prognosis in cytogenetically normal acute myeloid leukemia withNPM1 mutation without FLT3 internal tandem duplication,” J. Clin. Oncol28:3636-3643 (2010); Andersson, et al., “IDH1 and IDH2 mutations inpediatric acute leukemia,” Leukemia, 25(10):1-15 (2011)).

PIM kinases are a family of serine/threonine kinases composed of threedifferent isoforms (PIM1, PIM2, and PIM3). They differ partially intheir tissue distribution.

PIM1 is a proto-oncogene that encodes for serine or threonine kinases.In some cases, it has been described in relation to murine T-celllymphomas, but has since been found to be highly expressed in othertumor cells. PIM1 is involved in cell cycle progression, apoptosis,transcriptional activations, and signal transduction pathways.

PIM2 is a proto-oncogene that functions as a serine/threonine proteinkinase. PIM2 is involved in apoptosis, cell survival, and cellproliferation. It regulates MYC transcriptional activity, cell cycleprogression, and regulation of cap-dependent protein translation.Phosphorylation of MYC leads to an increase in MYC protein stability andthereby an increase in transcriptional activity. PIM2 regulatescap-dependent protein translation in a mammalian rapamycin complex 1(mTORC1)-independent manner and parallel to the PI3K-Akt pathway.

PIM3 is a proto-oncogene and functions as a serine/threonine proteinkinase. PIM3 is involved with apoptosis, cell survival, and proteintranslation. It also regulates MYC transcriptional activity.

Hematological Malignancies

Disclosed herein are methods of treating an individual having ahematological malignancy with a combination of a TEC inhibitor and ananticancer agent that inhibits MCL-1, IDH1, or MALT1. In someembodiments, the hematological malignancy is a leukemia, a lymphoma, amyeloma, a non-Hodgkin's lymphoma, a Hodgkin's lymphoma, T-cellmalignancy, or a B-cell malignancy.

In some embodiments, the hematological malignancy is a T-cellmalignancy. In some embodiments, T-cell malignancies include peripheralT-cell lymphoma not otherwise specified (PTCL-NOS), anaplastic largecell lymphoma, angioimmunoblastic lymphoma, cutaneous T-cell lymphoma,adult T-cell leukemia/lymphoma (ATLL), blastic NK-cell lymphoma,enteropathy-type T-cell lymphoma, hematosplenic gamma-delta T-celllymphoma, lymphoblastic lymphoma, nasal NK/T-cell lymphomas, ortreatment-related T-cell lymphomas.

In some embodiments, the hematological malignancy is a B-cellmalignancy. In some embodiments, B-cell malignancies include acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), acute monocytic leukemia (AMoL), chroniclymphocytic leukemia (CLL), high-risk chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma (SLL), high-risk small lymphocyticlymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma(DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia,multiple myeloma, extranodal marginal zone B cell lymphoma, nodalmarginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt highgrade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL),immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, Bcell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenicmarginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal(thymic) large B cell lymphoma, intravascular large B cell lymphoma,primary effusion lymphoma, or lymphomatoid granulomatosis. In someembodiments, the B-cell malignancy is diffuse large B-cell lymphoma(DLBCL). In some embodiments, the hematological malignancy is diffuselarge B-cell lymphoma (DLBCL). In some embodiments, the DLBCL is anactivated B-cell DLBCL (ABC-DLBCL), a germinal center B-cell like DLBCL(GBC-DLBCL), a double hit DLBCL (DH-DLBCL), or a triple hit DLBCL(TH-DLBCL).

In some embodiments, the hematological malignancy is a relapsed orrefractory hematological malignancy. In some embodiments, the relapsedor refractory hematological malignancy is a relapsed or refractoryT-cell malignancy. In some embodiments, the relapsed or refractoryhematological malignancy is a relapsed or refractory B-cell malignancy.In some embodiments, the relapsed or refractory B-cell malignancyinclude acute lymphoblastic leukemia (ALL), acute myelogenous leukemia(AML), chronic myelogenous leukemia (CML), acute monocytic leukemia(AMoL), chronic lymphocytic leukemia (CLL), high-risk chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risksmall lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuselarge B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom'smacroglobulinemia, multiple myeloma, extranodal marginal zone B celllymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma,non-Burkitt high grade B cell lymphoma, primary mediastinal B-celllymphoma (PMBL), immunoblastic large cell lymphoma, precursorB-lymphoblastic lymphoma, B cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cellmyeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma,intravascular large B cell lymphoma, primary effusion lymphoma, orlymphomatoid granulomatosis. In some embodiments, the relapsed orrefractory B-cell malignancy is diffuse large B-cell lymphoma (DLBCL).In some embodiments, the hematological malignancy is diffuse largeB-cell lymphoma (DLBCL). In some embodiments, the DLBCL is an activatedB-cell DLBCL (ABC-DLBCL), a germinal center B-cell like DLBCL(GBC-DLBCL), a double hit DLBCL (DH-DLBCL), a triple hit DLBCL(TH-DLBCL), or unclassified DLBCL. In some embodiments, the relapsed orrefractory hematological malignancy is diffuse large B-cell lymphoma(DLBCL).

In some embodiments, the hematological malignancy is a relapsedhematological malignancy. In some embodiments, the hematologicalmalignancy is a refractory hematological malignancy. In someembodiments, the refractory hematological malignancy contains anacquired resistance to a BTK inhibitor. In some embodiments, the BTKinhibitor is ibrutinib. In some embodiments, the refractoryhematological malignancy is BTK-resistant hematological malignancy. Insome embodiments, the hematological malignancy is BTK-resistanthematological malignancy.

DLBCL

Diffuse large B-cell lymphoma (DLBCL) refers to a neoplasm of thegerminal center B lymphocytes with a diffuse growth pattern and ahigh-intermediate proliferation index. DLBCLs represent approximately30% of all lymphomas and may present with several morphological variantsincluding the centroblastic, immunoblastic, T-cell/histiocyte rich,anaplastic and plasmoblastic subtypes. Genetic tests have shown thatthere are different subtypes of DLBCL. These subtypes seem to havedifferent outlooks (prognoses) and responses to treatment. DLBCL canaffect any age group but occurs mostly in older people (the average ageis mid-60s).

The ABC subtype of diffuse large B-cell lymphoma (ABC-DLBCL) is thoughtto arise from post germinal center B cells that are arrested duringplasmatic differentiation. The ABC subtype of DLBCL (ABC-DLBCL) accountsfor approximately 30% total DLBCL diagnoses. It is considered the leastcurable of the DLBCL molecular subtypes and, as such, patients diagnosedwith the ABC-DLBCL typically display significantly reduced survivalrates compared with individuals with other types of DLCBL. ABC-DLBCL ismost commonly associated with chromosomal translocations deregulatingthe germinal center master regulator BCL6 and with mutationsinactivating the PRDM1 gene, which encodes a transcriptional repressorrequired for plasma cell differentiation. In some embodiments, ABC-DLBCLcontains mutations within the cytoplasmic tails of the B cell receptorsubunits CD79A and CD79B. In some embodiments, the DLBCL containsmodifications in the PIM1, PIM2, and/or PIM3 genes. In some embodiments,the DLBCL contains modifications in the PIM1 gene. In some embodiments,the DLBCL contains modifications in the kinase domain of PIM1. In someembodiments, these modifications are mutations.

Disclosed herein, in certain embodiments, is a method for treating adiffuse large B-cell lymphoma (DLBCL) in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of a combination comprising a TEC inhibitor and an anticanceragent. In some embodiments, the TEC inhibitor is an ITK inhibitor or aBTK inhibitor. In some embodiments, disclosed herein is a method fortreating a diffuse large B-cell lymphoma (DLBCL) comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a combination comprising an ITK and an anticancer agent. Insome embodiments, disclosed herein is a method for treating a diffuselarge B-cell lymphoma (DLBCL) in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of acombination comprising a BTK and an anticancer agent. In someembodiments, the BTK inhibitor is selected from among ibrutinib(PCI-32765), PCI-45292, PCI-45466, AVL-101/CC-101 (AvilaTherapeutics/Celgene Corporation), AVL-263/CC-263 (AvilaTherapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited),LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences), ACP-196 (AcertaPharma) and JTE-051 (Japan Tobacco Inc). In some embodiments, the BTKinhibitor is ibrutinib. In some embodiments, disclosed herein is amethod for treating a diffuse large B-cell lymphoma (DLBCL) in a subjectin need thereof, comprising administering to the subject atherapeutically effective amount of a combination comprising ibrutiniband an anticancer agent. In some embodiments, the anticancer agent is aninhibitor of MCL-1, MALT1, IDH1, or JAK3. In some embodiments, theanticancer agent is an inhibitor of MCL-1, MALT1, or IDH1. In someembodiments, the MCL-1 inhbitor is selected from BI97C10, BI112D1,gossypol, obatoclax, MG-132, MIM1, sabutoclax, and TW-37. In someembodiments, the MALT1 inhibitor is selected from MI-2, mepazine,thioridazine, and promazine. In some embodiments, the IDH1 inhibitor isselected from AGI-5198, AG-120, IDH-C227, and ML309.

Disclosed herein, in certain embodiments, is a method for treatingdiffuse large B-cell lymphoma, activated B cell-like subtype (ABC-DLBCL)comprising administering to a subject in need thereof a therapeuticallyeffective amount of a combination comprising a TEC inhibitor and ananticancer agent. In some embodiments, the TEC inhibitor is an ITKinhibitor or a BTK inhibitor. In some embodiments, disclosed herein is amethod for treating ABC-DLBCL comprising administering to a subject inneed thereof a therapeutically effective amount of a combinationcomprising an ITK and an anticancer agent. In some embodiments,disclosed herein is a method for treating ABC-DLBCL comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a combination comprising a BTK and an anticancer agent. Insome embodiments, the BTK inhibitor is selected from among ibrutinib(PCI-32765), PCI-45292, PCI-45466, AVL-101/CC-101 (AvilaTherapeutics/Celgene Corporation), AVL-263/CC-263 (AvilaTherapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited),LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences), ACP-196 (AcertaPharma) and JTE-051 (Japan Tobacco Inc). In some embodiments, the BTKinhibitor is ibrutinib. In some embodiments, disclosed herein is amethod for treating ABC-DLBCL comprising administering to a subject inneed thereof a therapeutically effective amount of a combinationcomprising ibrutinib and an anticancer agent. In some embodiments, theanticancer agent is an inhibitor of MCL-1, MALT1, IDH1, or JAK3. In someembodiments, the anticancer agent is an inhibitor of MCL-1, MALT1, orIDH1. In some embodiments, the MCL-1 inhbitor is selected from BI97C10,BI112D1, gossypol, obatoclax, MG-132, MIM1, sabutoclax, and TW-37. Insome embodiments, the MALT1 inhibitor is selected from MI-2, mepazine,thioridazine, and promazine. In some embodiments, the IDH1 inhibitor isselected from AGI-5198, AG-120, IDH-C227, and ML309.

CLL/SLL

Chronic lymphocytic leukemia and small lymphocytic lymphoma (CLL/SLL)are commonly thought as the same disease with slightly differentmanifestations. Where the cancerous cells gather determines whether itis called CLL or SLL. When the cancer cells are primarily found in thelymph nodes, lima bean shaped structures of the lymphatic system (asystem primarily of tiny vessels found in the body), it is called SLL.SLL accounts for about 5% to 10% of all lymphomas. When most of thecancer cells are in the bloodstream and the bone marrow, it is calledCLL.

Both CLL and SLL are slow-growing diseases, although CLL, which is muchmore common, tends to grow slower. CLL and SLL are treated the same way.They are usually not considered curable with standard treatments, butdepending on the stage and growth rate of the disease, most patientslive longer than 10 years. Occasionally over time, these slow-growinglymphomas may transform into a more aggressive type of lymphoma.

Chronic lymphoid leukemia (CLL) is the most common type of leukemia. Itis estimated that 100,760 people in the United States are living with orare in remission from CLL. Most (>75%) people newly diagnosed with CLLare over the age of 50. Currently CLL treatment focuses on controllingthe disease and its symptoms rather than on an outright cure. CLL istreated by chemotherapy, radiation therapy, biological therapy, or bonemarrow transplantation. Symptoms are sometimes treated surgically(splenectomy removal of enlarged spleen) or by radiation therapy(“de-bulking” swollen lymph nodes). Though CLL progresses slowly in mostcases, it is considered generally incurable. Certain CLLs are classifiedas high-risk. As used herein, “high risk CLL” means CLL characterized byat least one of the following 1) 17p13-; 2) 11q22-; 3) unmutated IgVHtogether with ZAP-70+ and/or CD38+; or 4) trisomy 12.

CLL treatment is typically administered when the patient's clinicalsymptoms or blood counts indicate that the disease has progressed to apoint where it may affect the patient's quality of life.

Small lymphocytic leukemia (SLL) is very similar to CLL described supra,and is also a cancer of B-cells. In SLL the abnormal lymphocytes mainlyaffect the lymph nodes. However, in CLL the abnormal cells mainly affectthe blood and the bone marrow. The spleen may be affected in bothconditions. SLL accounts for about 1 in 25 of all cases of non-Hodgkinlymphoma. It can occur at any time from young adulthood to old age, butis rare under the age of 50. SLL is considered an indolent lymphoma.This means that the disease progresses very slowly, and patients tend tolive many years after diagnosis. However, most patients are diagnosedwith advanced disease, and although SLL responds well to a variety ofchemotherapy drugs, it is generally considered to be incurable. Althoughsome cancers tend to occur more often in one gender or the other, casesand deaths due to SLL are evenly split between men and women. Theaverage age at the time of diagnosis is 60 years.

Although SLL is indolent, it is persistently progressive. The usualpattern of this disease is one of high response rates to radiationtherapy and/or chemotherapy, with a period of disease remission. This isfollowed months or years later by an inevitable relapse. Re-treatmentleads to a response again, but again the disease will relapse. Thismeans that although the short-term prognosis of SLL is quite good, overtime, many patients develop fatal complications of recurrent disease.Considering the age of the individuals typically diagnosed with CLL andSLL, there is a need in the art for a simple and effective treatment ofthe disease with minimum side-effects that do not impede on thepatient's quality of life. The instant invention fulfills this longstanding need in the art.

Disclosed herein, in certain embodiments, is a method for treating CLLcomprising administering to a subject in need thereof a therapeuticallyeffective amount of a combination comprising a TEC inhibitor and ananticancer agent. In some embodiments, the TEC inhibitor is an ITKinhibitor or a BTK inhibitor. In some embodiments, disclosed herein is amethod for treating CLL comprising administering to a subject in needthereof a therapeutically effective amount of a combination comprisingan ITK and an anticancer agent. In some embodiments, disclosed herein isa method for treating CLL comprising administering to a subject in needthereof a therapeutically effective amount of a combination comprising aBTK and an anticancer agent. In some embodiments, the BTK inhibitor isselected from among ibrutinib (PCI-32765), PCI-45292, PCI-45466,AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation), AVL-263/CC-263(Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited),LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences), ACP-196 (AcertaPharma) and JTE-051 (Japan Tobacco Inc). In some embodiments, the BTKinhibitor is ibrutinib. In some embodiments, disclosed herein is amethod for treating CLL comprising administering to a subject in needthereof a therapeutically effective amount of a combination comprisingibrutinib and an anticancer agent. In some embodiments, the anticanceragent is an inhibitor of MCL-1, MALT1, IDH1, or JAK3. In someembodiments, the anticancer agent is an inhibitor of MCL-1, MALT1, orIDH1. In some embodiments, the MCL-1 inhbitor is selected from BI97C10,BI112D1, gossypol, obatoclax, MG-132, MIM1, sabutoclax, and TW-37. Insome embodiments, the MALT1 inhibitor is selected from MI-2, mepazine,thioridazine, and promazine. In some embodiments, the IDH1 inhibitor isselected from AGI-5198, AG-120, IDH-C227, and ML309.

Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell lymphomawith a poor prognosis. The overall survival for MCL patients is about 30to 43 months and fewer than 15% of the patients are long-term survivors.The average age of patients is in the early 60s. In some instances, menare often affected. The lymphoma is usually widespread when it isdiagnosed, involving lymph nodes, bone marrow, and, very often, thespleen. Mantle cell lymphoma is not a very fast growing lymphoma, but isdifficult to treat.

Only about 5% of lymphomas are of the MCL type. In some instances, MCLis further stratified based on its clinical course such as an indolentclinical course which is characterized by non-nodal leukemia disease, orblastoid and pleomorphic MCLs which are associated with advanced andaggressive disease.

MCL is characterized by a CD5 positive antigen-naive pregerminal centerB-cell within the mantle zone that surrounds normal germinal centerfollicles. MCL cells generally over-express cyclin D1 due to a t(11:14)chromosomal translocation in the DNA. More specifically, thetranslocation is at t(11;14)(q13;q32). In some instances, additionalcytogenetic abnormalities are present in MCL. In some instances, theadditional cytogenetic abnormalities include mutations within CARD11;MYC translocation and/or gene amplification; inactivation of cell cycleinhibitors p16/INK4A and p14/ARF; gains of 3q, 12q and losses of 9p, 9q,17p, 19p, and 6q24/25; mutations in TP53; truncation or missensemutations of within the PI3K domain of the ATM gene; and mutationswithin the NOTCH1 gene, which produces a C-terminal truncated proteinwith increased oncogenic activity; and/or mutations associated withCCND1, NOTCH2, BIRC3, WHSC1 (also known as MMSET or NSD2), MEF2B, TLR2,MLL2, PIM1, TAB2, CREBBP, ITK, MAP3K14, PLCγ2, TRAF3, mTOR, ERBB4,TNFRSF11A, REL, PRKCB, BCL2, CCND3, CD79A, MYD88, and NFKBIA.

In some embodiments, a mutation within CARD11 includes a mutation atamino acid residue position 116, 123, 176, 208, 223, 225, 233, 243, 244,331, 380, or 425, according to the CARD11 sequence as shown in Table 40.In some embodiments, a mutation within CARD11 includes G116S, F123I,M176L, K208M, D223N, L225LI, M233MGLNKM, S243P, L244P, D331G, D280V, orE425K. In some instances, a mutation within CARD11 is a mutation atamino acid residue position 225. In some instances, the mutation atamino acid residue position 225 is L225LI. In some embodiments, themutation at amino acid residue position is an insertion mutation. Insome instances, a triple adenine (A) base insertion occurs at nucleicacid position 675 as shown in Table 41. In some embodiments, the tripleA insertion at nucleic acid position 675 results in an amino acidmutation at position 225. In some embodiments, the triple A insertion atnucleic acid position 675 results in L225LI mutation. In someembodiments, the triple A insertion correspond to a triple thymine (T)insertion in its complementary DNA sequence. In some embodiments, thetriple T insertion is at position 675 in the complementary DNA sequenceto the nucleic acid sequence as shown in Table 41. In some instances,the triple T insertion results in an amino acid mutation at position225. In some embodiments, the triple A insertion results in L225LImutation.

As used herein, a mutation refers to an insertion, a substitution, adeletion, a missense mutation, or a combination thereof. In someembodiments, a mutation is a substitution. In some embodiments, amutation is an insertion. In some embodiments, the mutation is aninsertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50,or more nucleic acid residues. In some embodiments, the mutation is aninsertion of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50,or less nucleic acid residues.

In some embodiments, a mutation within Cyclin D1 (CCND1) includes amutation at amino acid residue position 47, 44, 290, 46, 42, or 41. Insome embodiments, a mutation within CCND1 includes C47S, Y44S, Y44Q,Y44D, V290G, K46E, V42E, or S41T.

Wolf-Hischhorn syndrome candidate 1 (WHSC1) encodes a histone 3methyltransferase of lysine-36 (H3K36). In some embodiments, WHSC1protein contains mutations at amino acid residue position 1099 and/or1150. In some embodiments, WHSC1 protein contains mutations E1099Kand/or T1150A.

Myeloid/lymphoid or mixed-lineage leukemia protein 2 (MLL2) is a histonemethyltransferase and in some instances, contains mutations in its FYRNand FYRC domains. In some embodiments, MLL2 protein contains mutationsat amino acid residue position 5272, 2771, 1724, 3604, 5225, and/or2839. In some embodiments, MLL2 protein contains mutations A5272P,R2771, D1724fs (frame shift), Q3604, R5225C, and/or 52839.

Myocyte enhancer factor 2B (MEF2B) is a member of the MADS/MEF2 familyof DNA binding proteins. In some embodiments, MEF2B protein containsmutations at amino acid residue position 23 and/or 49. In someembodiments, MEF2B protein contains mutations K23R and/or N49S.

The ATM serine/threonine kinase gene is involved in cellular developmentand DNA repair. In some embodiments, ATM protein contains mutations atamino acid residue position 1338, 323, 2730, 3008, 2526, 2437, 2727,1959, 2104, 2427, 2308, 2297, 2694, 148, 593, 1618, and/or 2489. In someembodiments, ATM protein contains mutations Q1448A, I323V, Q2730R,R3008C, R2526S, Y2437S, V2727A, E1959K, W2104, L2427L, A2308T, Q2297,G2694K, R248Q, T593fs (frame shift), R1618, and/or S2489F.

Baculoviral IAP repeat containing 3 (BIRC3) gene encodes a member of theIAP family of proteins that inhibit apoptosis by interaction with tumornecrosis factor receptor-associated factors TRAF1 and TRAF2. In someembodiments, BIRC3 protein contains mutations at amino acid residueposition 552, 560, 550, 575, 563, 591, 556, 600, and/or 557. In someembodiments, BIRC3 protein contains mutations Q552, C560Y, R550, L575V,K563, R591fs (frame shift), T556fs, R600G, and/or C557G.

Neurogenic locus notch homolog protein 2 (NOTCH2) is a type 1transmembrane protein that is involved in cellular development. In someembodiments, NOTCH2 protein contains mutations at amino acid residueposition 2400, 2360, 2293, 2292, 2391, and/or 2285. In some embodiments,NOTCH2 protein contains mutations R2400, Q2360, H2293fs (frame shift),K2292fs, S2391fs, and/or Q2285.

In some embodiments, NOTCH1 protein contains mutations at amino acidresidue position 2515, 2504, 2281, 2487, and/or 2428. In someembodiments, NOTCH1 protein contains mutations P2515fs (frame shift),V2504fs, G2281fs, Q2487, and/or H2428fs.

In some embodiments, TLR2 protein contains mutations at amino acidresidue position 327 and/or 298. In some embodiments, TLR2 proteincontains mutations D327V and/or Y298S.

In some instances, MCL is characterized with the translocation att(11;14)(q13;q32) and with one or more of the additional cytogeneticabnormalities. In some instances, MCL is characterized with one or moreof the additional cytogenetic abnormalities but without thetranslocation at t(11;14)(q13;q32).

In some instances, MCL is characterized with an over-expression ofcyclin D1 and with one or more of the additional cytogeneticabnormalities. In some instances, MCL is characterized with one or moreof the additional cytogenetic abnormalities but without theover-expression of cyclin D1.

In some embodiments, MCL is characterized with the translocation att(11;14)(q13;q32) and with a mutation in CARD11. In some embodiments,the CARD11 mutation is a mutation at amino acid residue position 116,123, 176, 208, 223, 225, 233, 243, 244, 331, 380, or 425, according tothe CARD11 sequence as shown in Table 40. In some embodiments, theCARD11 mutation includes G116S, F123I, M176L, K208M, D223N, L225LI,M233MGLNKM, S243P, L244P, D331G, D280V, or E425K. In some embodiments,MCL is characterized with a mutation in CARD11 but without thetranslocation at t(11;14)(q13;q32).

In some embodiments, MCL is characterized with an over-expression ofcyclin D1 and with a mutation in CARD11. In some embodiments, the CARD11mutation is a mutation at amino acid residue position 116, 123, 176,208, 223, 225, 233, 243, 244, 331, 380, or 425, according to the CARD11sequence as shown in Table 40. In some embodiments, the CARD11 mutationincludes G116S, F123I, M176L, K208M, D223N, L225LI, M233MGLNKM, S243P,L244P, D331G, D280V, or E425K. In some embodiments, MCL is characterizedwith a mutation in CARD11 but without the over-expression of cyclin D1.

In some embodiments, MCL is characterized with the translocation att(11;14)(q13;q32), with a mutation in CARD11, and one or more mutationssuch as a mutation in BTK (e.g. C481S) and/or PLCγ2 mutations (e.g.R665W, S707F, and/or L845F). In some embodiments, the CARD11 mutation isa mutation at amino acid residue position 116, 123, 176, 208, 223, 225,233, 243, 244, 331, 380, or 425, according to the CARD11 sequence asshown in Table 40. In some embodiments, the CARD11 mutation includesG116S, F123I, M176L, K208M, D223N, L225LI, M233MGLNKM, S243P, L244P,D331G, D280V, or E425K. In some embodiments, MCL is characterized with amutation in CARD11 and one or more mutations such as a mutation in BTK(e.g. C481S) and/or PLCγ2 mutations (e.g. R665W, S707F, and/or L845F)but without the translocation at t(11;14)(q13;q32). In some embodiments,MCL is characterized with a mutation in CARD11 but without the one ormore mutations such as a mutation in BTK (e.g. C481S) and/or PLCγ2mutations (e.g. R665W, S707F, and/or L845F) and without thetranslocation at t(11;14)(q13;q32).

In some embodiments, MCL is characterized with an over-expression ofcyclin D1, with a mutation in CARD11, and one or more mutations such asa mutation in BTK (e.g. C481S) and/or PLCγ2 mutations (e.g. R665W,S707F, and/or L845F). In some embodiments, the CARD11 mutation is amutation at amino acid residue position 116, 123, 176, 208, 223, 225,233, 243, 244, 331, 380, or 425, according to the CARD11 sequence asshown in Table 40. In some embodiments, the CARD11 mutation includesG116S, F123I, M176L, K208M, D223N, L225LI, M233MGLNKM, S243P, L244P,D331G, D280V, or E425K. In some embodiments, MCL is characterized with amutation in CARD11 and one or more mutations such as a mutation in BTK(e.g. C481S) and/or PLCγ2 mutations (e.g. R665W, S707F, and/or L845F)but without the over-expression of cyclin D1. In some embodiments, MCLis characterized with a mutation in CARD11 but without one or moremutations such as a mutation in BTK (e.g. C481S) and/or PLCγ2 mutations(e.g. R665W, S707F, and/or L845F) and without the over-expression ofcyclin D1.

In some embodiments, MCL is a primary resistant MCL. In someembodiments, MCL is characterized with mutations in PIM1, TAB2, WHSC1,CREBBP, MLL2, ITK, MAP3K14, MYC, PLCγ2, TRAF3, mTOR, ERBB4, TNFRSF11A,REL, PRKCB, BCL2, CCND3, CD79A, MYD88, and NFKBIA. In some embodiments,a primary resistant MCL is characterized with mutations in TAB2, WHSC1,CREBBP, MLL2, ITK, MAP3K14, MYC, PLCγ2, TRAF3, mTOR, ERBB4, TNFRSF11A,REL, PRKCB, BCL2, CCND3, CD79A, MYD88, and NFKBIA. In some embodiments,patients who are primary resistant to MCL have mutations in PIM1, TAB2,WHSC1, CREBBP, MLL2, ITK, MAP3K14, MYC, PLCγ2, TRAF3, mTOR, ERBB4,TNFRSF11A, REL, PRKCB, BCL2, CCND3, CD79A, MYD88, and NFKBIA.

In some embodiments, patients with moderate clinical benefit havemutations in PIM1, TAB2, WHSC1, CREBBP, MLL2, ITK, MAP3K14, MYC, PLCγ2,TRAF3, mTOR, ERBB4, TNFRSF11A, REL, PRKCB, BCL2, CCND3, CD79A, MYD88,and NFKBLIA.

In some embodiments, patients with durable responses have mutations inTAB2, WHSC1, CREBBP, MLL2, ITK, MAP3K14, MYC, PLCγ2, TRAF3, mTOR, ERBB4,TNFRSF11A, REL, PRKCB, BCL2, CCND3, CD79A, MYD88, and NFKBIA. In someinstances, patients with durable responses have mutations in CREBBP,MLL2, mTOR, ERBB4, and TNFRSF11A.

Disclosed herein, in certain embodiments, is a method for treatingmantle cell lymphoma comprising administering to a subject in needthereof a therapeutically effective amount of a combination comprising aTEC inhibitor and an anticancer agent. In some embodiments, the TECinhibitor is an ITK inhibitor or a BTK inhibitor. In some embodiments,disclosed herein is a method for treating mantle cell lymphomacomprising administering to a subject in need thereof a therapeuticallyeffective amount of a combination comprising an ITK and an anticanceragent. In some embodiments, disclosed herein is a method for treatingmantle cell lymphoma comprising administering to a subject in needthereof a therapeutically effective amount of a combination comprising aBTK and an anticancer agent. In some embodiments, the BTK inhibitor isselected from among ibrutinib (PCI-32765), PCI-45292, PCI-45466,AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation), AVL-263/CC-263(Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited),LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences), ACP-196 (AcertaPharma), and JTE-051 (Japan Tobacco Inc). In some embodiments, the BTKinhibitor is ibrutinib. In some embodiments, disclosed herein is amethod for treating mantle cell lymphoma comprising administering to asubject in need thereof a therapeutically effective amount of acombination comprising ibrutinib and an anticancer agent. In someembodiments, the anticancer agent is an inhibitor of MCL-1, MALT1, IDH1,JAK3, proteasome, or PIM1. In some embodiments, the anticancer agent isan inhibitor of MCL-1, MALT1, IDH1, proteasome or PIM1. In someembodiments, the MCL-1 inhibitor is selected from BI97C10, BI112D1,gossypol, obatoclax, MG-132, MIM1, sabutoclax, and TW-37. In someembodiments, the MALT1 inhibitor is selected from MI-2, mepazine,thioridazine, and promazine. In some embodiments, the IDH1 inhibitor isselected from AGI-5198, AG-120, IDH-C227, and ML309. In someembodiments, the proteasome inhibitor is selected from carfilzomib andvelcade. In some embodiments, the PIM1 inhibitor is selected frommitoxantrone, SGI-1776, AZD1208, AZD1897, LGH447, JP_11646, Pim1inhibitor 2, SKI-O-068, CX-6258, AR460770, AR00459339 (Array BiopharmaInc.), miR-33a, Pim-1 inhibitory p27 (Kip1) peptide, LY333′531, K00135,quercetagein (3,3′,4′,5,6,7-hydroxyflavone), and LY294002. In someembodiments, MCL contains one or more cytogenetic abnormalities (e.g.translocation at t(11;14)(q13;q32) leading to over-expression of cyclinD1, CARD11, MYC translocation and/or gene amplification, or the like).In some embodiments, MCL contains a CARD11 mutation. In someembodiments, the CARD11 mutation is a mutation at amino acid residueposition 116, 123, 176, 208, 223, 225, 233, 243, 244, 331, 380, or 425,according to the CARD11 sequence as shown in Table 40. In someembodiments, the CARD11 mutation includes G116S, F123I, M176L, K208M,D223N, L225LI, M233MGLNKM, S243P, L244P, D331G, D280V, or E425K. In someembodiments, the CARD11 mutation is L225LI. In some embodiments, MCLcontains a CARD11 mutation and one or more additional cytogeneticabnormalities. In some embodiments, MCL contains a CARD11 mutation andone or more mutations such as a mutation in BTK (e.g. C481S) and/orPLCγ2 mutations (e.g. R665W, S707F, and/or L845F). In some embodiments,MCL contains a CARD11 mutation but does not contain one or moremutations such as a mutation in BTK (e.g. C481S) and/or PLCγ2 mutations(e.g. R665W, S707F, and/or L845F). In some embodiments, MCL is anibrutinib-resistant MCL. In some embodiments, MCL is a primary resistantMCL. In some embodiments, MCL has one or more mutations as shown in FIG.11. In some embodiments, primary resistant MCL has one or more mutationsas shown in FIG. 11.

Waldenstrom's Macroglobulinemia

Waldenstrom's macroglobulinemia, also known as lymphoplasmacyticlymphoma, is cancer involving a subtype of white blood cells calledlymphocytes. It is characterized by an uncontrolled clonal proliferationof terminally differentiated B lymphocytes. It is also characterized bythe lymphoma cells making an antibody called immunoglobulin M (IgM). TheIgM antibodies circulate in the blood in large amounts, and cause theliquid part of the blood to thicken, like syrup. This can lead todecreased blood flow to many organs, which can cause problems withvision (because of poor circulation in blood vessels in the back of theeyes) and neurological problems (such as headache, dizziness, andconfusion) caused by poor blood flow within the brain. Other symptomscan include feeling tired and weak, and a tendency to bleed easily. Theunderlying etiology is not fully understood but a number of risk factorshave been identified, including the locus 6p21.3 on chromosome 6. Thereis a 2- to 3-fold risk increase of developing WM in people with apersonal history of autoimmune diseases with autoantibodies andparticularly elevated risks associated with hepatitis, humanimmunodeficiency virus, and rickettsiosis.

Disclosed herein, in certain embodiments, is a method for treatingWaldenstrom's macroglobulinemia comprising administering to a subject inneed thereof a therapeutically effective amount of a combinationcomprising a TEC inhibitor and an anticancer agent. In some embodiments,the TEC inhibitor is an ITK inhibitor or a BTK inhibitor. In someembodiments, disclosed herein is a method for treating Waldenstrom'smacroglobulinemia comprising administering to a subject in need thereofa therapeutically effective amount of a combination comprising an ITKand an anticancer agent. In some embodiments, disclosed herein is amethod for treating Waldenstrom's macroglobulinemia comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a combination comprising a BTK and an anticancer agent. Insome embodiments, the BTK inhibitor is selected from among ibrutinib(PCI-32765), PCI-45292, PCI-45466, AVL-101/CC-101 (AvilaTherapeutics/Celgene Corporation), AVL-263/CC-263 (AvilaTherapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited),LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences), ACP-196 (AcertaPharma) and JTE-051 (Japan Tobacco Inc). In some embodiments, the BTKinhibitor is ibrutinib. In some embodiments, disclosed herein is amethod for treating Waldenstrom's macroglobulinemia comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a combination comprising ibrutinib and an anticancer agent. Insome embodiments, the anticancer agent is an inhibitor of MCL-1, MALT1,IDH1, or JAK3. In some embodiments, the anticancer agent is an inhibitorof MCL-1, MALT1, or IDH1. In some embodiments, the MCL-1 inhbitor isselected from BI97C10, BI112D1, gossypol, obatoclax, MG-132, MIM1,sabutoclax, and TW-37. In some embodiments, the MALT1 inhibitor isselected from MI-2, mepazine, thioridazine, and promazine. In someembodiments, the IDH1 inhibitor is selected from AGI-5198, AG-120,IDH-C227, and ML309.

TEC Family Kinase Inhibitors

BTK is a member of the Tyrosine-protein kinase (TEC) family of kinases.In some embodiments, the TEC family comprises BTK, ITK, TEC, RLK andBMX. In some embodiments, a covalent TEC family kinase inhibitorinhibits the kinase activity of BTK, ITK, TEC, RLK and BMX. In someembodiments, a covalent TEC family kinase inhibitor is a BTK inhibitor.In some embodiments, a covalent TEC family kinase inhibitor is an ITKinhibitor. In some embodiments, a covalent TEC family kinase inhibitoris a TEC inhibitor. In some embodiments, a covalent TEC family kinaseinhibitor is a RLK inhibitor. In some embodiments, a covalent TEC familykinase inhibitor is a BMK inhibitor.

BTK Inhibitor Compounds Including Ibrutinib, and PharmaceuticallyAcceptable Salts Thereof

The BTK inhibitor compound described herein (i.e. ibrutinib) isselective for BTK and kinases having a cysteine residue in an amino acidsequence position of the tyrosine kinase that is homologous to the aminoacid sequence position of cysteine 481 in BTK. The BTK inhibitorcompound can form a covalent bond with Cys 481 of BTK (e.g., via aMichael reaction).

In some embodiments, the BTK inhibitor is a compound of Formula (A)having the structure:

wherein:

A is N;

R₁ is phenyl-O-phenyl or phenyl-S-phenyl;

R₂ and R₃ are independently H;

R₄ is L₃-X-L₄-G, wherein,

L₃ is optional, and when present is a bond, optionally substituted orunsubstituted alkyl, optionally substituted or unsubstituted cycloalkyl,optionally substituted or unsubstituted alkenyl, optionally substitutedor unsubstituted alkynyl;

X is optional, and when present is a bond, —O—, —C(═O)—, —S—, —S(═O)—,—S(═O)₂—, —NH—, —NR₉—, —NHC(O)—, —C(O)NH—, —NR₉C(O)—, —C(O)NR₉—,—S(═O)₂NH—, —NHS(═O)₂—, —S(═O)₂NR₉—, —NR₉S(═O)₂—, —OC(O)NH—, —NHC(O)O—,—OC(O)NR₉—, —NR₉C(O)O—, —CH═NO—, —ON═CH—, —NR₁₀C(O)NR₁₀—, heteroaryl-,aryl-, —NR₁₀C(═NR₁₁)NR₁₀—, —NR₁₀C(═NR₁₁)—, —C(═NR₁₁)NR₁₀—, —OC(═NR₁₁)—,or —C(═NR₁₁)O—;

L4 is optional, and when present is a bond, substituted or unsubstitutedalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,substituted or unsubstituted heterocycle;

or L3, X and L4 taken together form a nitrogen containing heterocyclicring;

G is

wherein,

R₆, R₇ and R₈ are independently selected from among H, halogen, CN, OH,substituted or unsubstituted alkyl or substituted or unsubstitutedheteroalkyl or substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl;

each R₉ is independently selected from among H, substituted orunsubstituted lower alkyl, and substituted or unsubstituted lowercycloalkyl;

each R₁₀ is independently H, substituted or unsubstituted lower alkyl,or substituted or unsubstituted lower cycloalkyl; or

two R₁₀ groups can together form a 5-, 6-, 7-, or 8-memberedheterocyclic ring; or

R₁₀ and R₁₁ can together form a 5-, 6-, 7-, or 8-membered heterocyclicring; or each R₁₁ is independently selected from H or substituted orunsubstituted alkyl; or a pharmaceutically acceptable salt thereof. Insome embodiments, L₃, X and L₄ taken together form a nitrogen containingheterocyclic ring. In some embodiments, the nitrogen containingheterocyclic ring is a piperidine group. In some embodiments, G is

In some embodiments, the compound of Formula (A) is1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one.

“Ibrutinib” or“1-((R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one”or“1-{(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl}prop-2-en-1-one”or “2-Propen-1-one,1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidinyl-”or Ibrutinib or any other suitable name refers to the compound with thefollowing structure:

A wide variety of pharmaceutically acceptable salts is formed fromIbrutinib and includes:

-   -   acid addition salts formed by reacting Ibrutinib with an organic        acid, which includes aliphatic mono- and dicarboxylic acids,        phenyl-substituted alkanoic acids, hydroxyl alkanoic acids,        alkanedioic acids, aromatic acids, aliphatic and aromatic        sulfonic acids, amino acids, etc. and include, for example,        acetic acid, trifluoroacetic acid, propionic acid, glycolic        acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,        succinic acid, fumaric acid, tartaric acid, citric acid, benzoic        acid, cinnamic acid, mandelic acid, methanesulfonic acid,        ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and        the like;    -   acid addition salts formed by reacting Ibrutinib with an        inorganic acid, which includes hydrochloric acid, hydrobromic        acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic        acid, hydrofluoric acid, phosphorous acid, and the like.

The term “pharmaceutically acceptable salts” in reference to Ibrutinibrefers to a salt of Ibrutinib, which does not cause significantirritation to a mammal to which it is administered and does notsubstantially abrogate the biological activity and properties of thecompound.

It should be understood that a reference to a pharmaceuticallyacceptable salt includes the solvent addition forms (solvates). Solvatescontain either stoichiometric or non-stoichiometric amounts of asolvent, and are formed during the process of product formation orisolation with pharmaceutically acceptable solvents such as water,ethanol, methanol, methyl tert-butyl ether (MTBE), diisopropyl ether(DIPE), ethyl acetate, isopropyl acetate, isopropyl alcohol, methylisobutyl ketone (MIBK), methyl ethyl ketone (MEK), acetone,nitromethane, tetrahydrofuran (THF), dichloromethane (DCM), dioxane,heptanes, toluene, anisole, acetonitrile, and the like. In one aspect,solvates are formed using, but limited to, Class 3 solvent(s).Categories of solvents are defined in, for example, the InternationalConference on Harmonization of Technical Requirements for Registrationof Pharmaceuticals for Human Use (ICH), “Impurities: Guidelines forResidual Solvents, Q3C(R3), (November 2005). Hydrates are formed whenthe solvent is water, or alcoholates are formed when the solvent isalcohol. In some embodiments, solvates of Ibrutinib, or pharmaceuticallyacceptable salts thereof, are conveniently prepared or formed during theprocesses described herein. In some embodiments, solvates of Ibrutinibare anhydrous. In some embodiments, Ibrutinib, or pharmaceuticallyacceptable salts thereof, exist in unsolvated form. In some embodiments,Ibrutinib, or pharmaceutically acceptable salts thereof, exist inunsolvated form and are anhydrous.

In yet other embodiments, Ibrutinib, or a pharmaceutically acceptablesalt thereof, is prepared in various forms, including but not limitedto, amorphous phase, crystalline forms, milled forms andnano-particulate forms. In some embodiments, Ibrutinib, or apharmaceutically acceptable salt thereof, is amorphous. In someembodiments, Ibrutinib, or a pharmaceutically acceptable salt thereof,is amorphous and anhydrous. In some embodiments, Ibrutinib, or apharmaceutically acceptable salt thereof, is crystalline. In someembodiments, Ibrutinib, or a pharmaceutically acceptable salt thereof,is crystalline and anhydrous.

In some embodiments, Ibrutinib is prepared as outlined in U.S. Pat. No.7,514,444.

In some embodiments, the Btk inhibitor is PCI-45292, PCI-45466,AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation), AVL-263/CC-263(Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited),LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences), ACP-196 (AcertaPharma) or JTE-051 (Japan Tobacco Inc).

In some embodiments, the BTK inhibitor is4-(tert-butyl)-N-(2-methyl-3-(4-methyl-6-((4-(morpholine-4-carbonyl)phenyl)amino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)benzamide(CGI-1746);7-benzyl-1-(3-(piperidin-1-yl)propyl)-2-(4-(pyridin-4-yl)phenyl)-1H-imidazo[4,5-g]quinoxalin-6(5H)-one(CTA-056);(R)—N-(3-(6-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide(GDC-0834);6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-1-one(RN-486);N-[5-[5-(4-acetylpiperazine-1-carbonyl)-4-methoxy-2-methylphenyl]sulfanyl-1,3-thiazol-2-yl]-4-[(3,3-dimethylbutan-2-ylamino)methyl]benzamide(BMS-509744, HY-11092); orN-(5-45-(4-Acetylpiperazine-1-carbonyl)-4-methoxy-2-methylphenyl)thio)thiazol-2-yl)-4-(((3-methylbutan-2-yl)amino)methyl)benzamide(HY11066); or a pharmaceutically acceptable salt thereof.

In some embodiments, the BTK inhibitor is:

or a pharmaceutically acceptable salt thereof.

ITK Inhibitors

In some embodiments, the ITK inhibitor covalently binds to Cysteine 442of ITK. In some embodiments, the ITK inhibitor is an ITK inhibitorcompound described in WO2002/0500071, which is incorporated by referencein its entirety. In some embodiments, the ITK inhibitor is an ITKinhibitor compound described in WO2005/070420, which is incorporated byreference in its entirety. In some embodiments, the ITK inhibitor is anITK inhibitor compound described in WO2005/079791, which is incorporatedby reference in its entirety. In some embodiments, the ITK inhibitor isan ITK inhibitor compound described in WO2007/076228, which isincorporated by reference in its entirety. In some embodiments, the ITKinhibitor is an ITK inhibitor compound described in WO2007/058832, whichis incorporated by reference in its entirety. In some embodiments, theITK inhibitor is an ITK inhibitor compound described in WO2004/016610,which is incorporated by reference in its entirety. In some embodiments,the ITK inhibitor is an ITK inhibitor compound described inWO2004/016611, which is incorporated by reference in its entirety. Insome embodiments, the ITK inhibitor is an ITK inhibitor compounddescribed in WO2004/016600, which is incorporated by reference in itsentirety. In some embodiments, the ITK inhibitor is an ITK inhibitorcompound described in WO2004/016615, which is incorporated by referencein its entirety. In some embodiments, the ITK inhibitor is an ITKinhibitor compound described in WO2005/026175, which is incorporated byreference in its entirety. In some embodiments, the ITK inhibitor is anITK inhibitor compound described in WO2006/065946, which is incorporatedby reference in its entirety. In some embodiments, the ITK inhibitor isan ITK inhibitor compound described in WO2007/027594, which isincorporated by reference in its entirety. In some embodiments, the ITKinhibitor is an ITK inhibitor compound described in WO2007/017455, whichis incorporated by reference in its entirety. In some embodiments, theITK inhibitor is an ITK inhibitor compound described in WO2008/025820,which is incorporated by reference in its entirety. In some embodiments,the ITK inhibitor is an ITK inhibitor compound described inWO2008/025821, which is incorporated by reference in its entirety. Insome embodiments, the ITK inhibitor is an ITK inhibitor compounddescribed in WO2008/025822, which is incorporated by reference in itsentirety. In some embodiments, the ITK inhibitor is an ITK inhibitorcompound described in WO2011/017219, which is incorporated by referencein its entirety. In some embodiments, the ITK inhibitor is an ITKinhibitor compound described in WO2011/090760, which is incorporated byreference in its entirety. In some embodiments, the ITK inhibitor is anITK inhibitor compound described in WO2009/158571, which is incorporatedby reference in its entirety. In some embodiments, the ITK inhibitor isan ITK inhibitor compound described in WO2009/051822, which isincorporated by reference in its entirety. In some embodiments, the ITKinhibitor is an ITK inhibitor compound described in US 20110281850,which is incorporated by reference in its entirety. In some embodiments,the Itk inhibitor is an Itk inhibitor compound described inWO2014/082085, which is incorporated by reference in its entirety. Insome embodiments, the Itk inhibitor is an Itk inhibitor compounddescribed in WO2014/093383, which is incorporated by reference in itsentirety. In some embodiments, the Itk inhibitor is an Itk inhibitorcompound described in U.S. Pat. No. 8,759,358, which is incorporated byreference in its entirety. In some embodiments, the Itk inhibitor is anItk inhibitor compound described in WO2014/105958, which is incorporatedby reference in its entirety. In some embodiments, the Itk inhibitor isan Itk inhibitor compound described in US2014/0256704, which isincorporated by reference in its entirety. In some embodiments, the Itkinhibitor is an Itk inhibitor compound described in US20140315909, whichis incorporated by reference in its entirety. In some embodiments, theItk inhibitor is an Itk inhibitor compound described in US20140303161,which is incorporated by reference in its entirety. In some embodiments,the Itk inhibitor is an Itk inhibitor compound described inWO2014/145403, which is incorporated by reference in its entirety.

In some embodiments, the ITK inhibitor has a structure selected from:

Anticancer Agents MALT1 Inhibitors

Disclosed herein, in certain embodiments, are MALT1 inhibitors incombination with a BTK inhibitor for the treatment of a hematologicalmalignancy. In some embodiments, the MALT1 inhibitors include, but arenot limited to MI-2 and phenothiazine derivatives such as mepazine,thioridazine, and promazine (see, Nagel et al., “Pharmacologicinhibition of MALT1 protease by phenothiazines as a therapeutic approachfor the treatment of aggressive ABC-DLBCL,” Cell 22:825-837 (2012)). Insome embodiments, a MALT1 inhibitor is a MALT1 inhibitor disclosed inFontan et al, “MALT1 small molecule inhibitors specifically suppressABC_DLBCL in vitro and in vivo,” Cell 22:812-824 (2012). In someembodiments, a MALT1 inhibitor is a MALT1 inhibitor disclosed in any ofthe following patent publications: WO2013017637; WO2014086478;WO2014074815; and U.S. Pat. No. 8,309,523.

JAK3 Inhibitors

Disclosed herein, in certain embodiments, are JAK3 inhibitors incombination with a BTK inhibitor for the treatment of a hematologicalmalignancy. In some embodiments, the JAK3 inhibitors include, but arenot limited to, AT9283, benzoxathiol derivatives such as BOT-4-one,cercosporamide, JAK3 Inhibitor IV, JAK3 Inhibitor V, JAK3 Inhibitor VI,JAK3 Inhibitor VII, JANEX-1, MS-1020, PF-956980 (Pfizer), ruxolitinib,TCS21311, TG101209, tofacitinib (tasocitinib; CP-690550; Xeljanz andJakvinus, Pfizer), VX-509 (Vertex Pharmaceuticals Inc.), WHI-P 131, andWHI-P 154. In some embodiments, a JAK3 inhibitor is a JAK3 inhibitordisclosed in Chen, et al., “Development of pyrimidine-based inhibitorsof Janus tyrosine kinase 3,” Bioorg Med Chem Lett 16(21):5633-5638(2006); Brown, et al., “Naphthyl ketones: new class of Janus kinase 3inhibitors,” Bioorg Med Chem Lett 10(6):575-579 (2000); Jaime-Figueroa,et al., “Discovery of a series of novel5H-pyrrolo[2,3-b]pyrazine-2-phenyl ethers, as potent JAK3 kinaseinhibitors,” Bioorg Med Chem Lett 23(9):2522-2526 (2013); Cole, et al.,“2-Benzimidazolyl-9-(chroman-4-yl)-purinone derivatives as JAK3inhibitors,” Bioorg Med Chem Lett 19(23):6788-6792 (2009); and Clark etal., “Development of new pyrrolopyrimidine-based inhibitors of Januskinase 3 (JAK3),” Bioorg Med Chem Lett 17(5):1250-1253 (2007).

In some embodiments, a JAK3 inhibitor is a JAK3 inhibitor disclosed inany of the following patent publications: WO2014081732; WO2014039595;WO2000051587; WO2012143320; WO2010118986; WO2012046793; WO2010014930;WO2004099204; WO2005075429; WO2011051452; WO2008119792; WO2008148867;WO2008119792; WO2008060301; WO2010039518; US2010009978; US2010239631;and US2010210623.

MCL-1 Inhibitors

Disclosed herein, in certain embodiments, are MCL-1 inhibitors incombination with a BTK inhibitor for the treatment of a hematologicalmalignancy. In some embodiments, the MCL-1 inhibitors include, but arenot limited to BI97C10, BI112D1, gossypol (AT-101, AscentaTherapeutics), obatoclax (GX15-070, Cephalon), MG-132, MIM1, sabutoclax(BI97C1, Oncothyreon), and TW-37. In some embodiments, a MCL-1 inhibitoris a MCL-1 inhibitor disclosed in Varadarajan, et al., “Evaluation andcritical assessment of putative MCL-1 inhibitors,” Cell Death &Differentiation 20:1475-1484 (2013); Tanaka, et al., “Discovery ofpotent Mcl-1/Bcl-xL dual inhibitors by using a hybridization strategybased on structural analysis of target proteins,” J Med Chem56(23):9635-9645 (2013); and Friberg, et al., “Discovery of potentmyeloid cell leukemia 1 (Mcl 1) inhibitors using fragment based methodsand structure based design,” J Med Chem 56(1):15-30 (2013).

In some embodiments, a MCL-1 inhibitor is a MCL-1 inhibitor disclosed inany of the following patent publications: WO2013052943; WO2013149124;WO2013142281; WO2011094708; WO2013112878; WO2008131000; WO2014047427;CN101352437; and US20110112112.

IDH1 Inhibitors

Disclosed herein, in certain embodiments, are IDH1 inhibitors incombination with a BTK inhibitor for the treatment of a hematologicalmalignancy. In some embodiments, the IDH1 inhibitors include, but arenot limited to AGI-5198, AG-120 (Agios Pharmaceuticals, Inc.), IDH-C227(Agios Pharmaceuticals, Inc.), and ML309 (Agios Pharmaceuticals, Inc.).In some embodiments, an IDH1 inhibitor is an IDH1 inhibitor disclosed inDavis, et al. ML309: A potent inhibitor of R132H mutant IDH1 capable ofreducing 2-hydroxyglutarate production in U87 MG glioblastoma cells.2012 Apr. 16 [Updated 2013 May 8]. In: Probe Reports from the NIHMolecular Libraries Program [Internet]. Bethesda (Md.): National Centerfor Biotechnology Information (US); 2010; Popovici-Muller, et al.,“Discovery of the first potent inhibitors of mutant IDH1 that lowertumor 2-HG in vivo,” ACS Med Chem Lett 3(10):850-855 (2012).

In some embodiments, an IDH1 inhibitor is an IDH1 inhibitor disclosed inany of the following patent publications: WO2014062511; WO2012171506;WO2012171337; WO2013107405; WO2013107291; WO2012009678; andWO2011072174.

Proteasome Inhibitors

Disclosed herein, in certain embodiments, are proteasome inhibitors incombination with a BTK inhibitor for the treatment of a hematologicalmalignancy. In some embodiments, the proteasome inhibitors include, butare not limited to, carfilzomib (ONYX), bortezomib (Velcade,Millennium), disulfiram, epigallocatechin-3-gallate, marizomib (Nereus),NPI-0052, MLN9708 (Millennium), CEP-18770 (Cephalon), ONX 0912 (ONYX),salinosporamide A, epoxomicin, MG132, PSI, fellutamide B, MLN2238,MLN9708, omuralide, PS-519, belactosin A, ¹²⁵I-NIP-L3VS, MV151, SylA,GlbA, HT1171, GLS, TMC95A, Argyrin A, scytonemide A, ritonavir,benzylstatine peptide 1, capped dipeptide 1, capped dipeptide 2,CVT-659, PI-083, and hydroxyurea inhibitor.

PIM Inhibitors

Disclosed herein, in certain embodiments, are PIM inhibitors incombination with a BTK inhibitor for the treatment of a hematologicalmalignancy. As used herein, “PIM inhibitor(s)” may be “pan-PIMinhibitor.” “PIM inhibitor(s) may aslo be “PIM1 inhibitors.”Accordingly, in some embodiments, a “PIM inhibitor” refers to aninhibitor of PIM1. In some embodiments, “PIM inhibitor” refers to a“pan-PIM inhibitor,” or an inhibitor of PIM1, PIM2, and PIM3. PIMinhibitors may also be referred to as PIM kinase inhibitors. ExemplaryPIM inhibitors include, but are not limited to, mitoxantrone, SGI-1776,AZD1208, AZD1897, LGH447, JP_11646, Pim1 inhibitor 2, SKI-O-068,CX-6258, AR460770, AR00459339 (Array Biopharma Inc.), miR-33a, Pim-1inhibitory p27 (Kip1) peptide, LY333′531, K00135, quercetagein(3,3′,4′,5,6,7-hydroxyflavone), and LY294002. In some embodiments, thePIM inhibitor is AZD1208.

In some embodiments, PIM1 inhibitors include rucaparib and veliparib asdescribed in Antolin, et al., “Linking off-target kinase pharmacology tothe differential cellular effects observed among PARP inhibitors,”Oncotarget 5(10):3023-3028 (2014); pyrrolo[1,2-a]pyrazinones asdescribed in Casuscelli et al., “Discovery and optimization ofpyrrolo[1,2-a]pyrazinones leads to novel and selective inhibitors of PIMkinases,”Bioorg Med Chem. 21(23):7364-7380 (2013); as described inYoshida et al., “Synthesis, resolution, and biological evaluation ofatropisomeric (aR)- and (aS)-16-methyllamellarins N: unique effects ofthe axial chirality on the selectivity of protein kinases inhibition,” JMed Chem 56(18):7289-7301 (2013); as described in Cozza et al.,“Exploiting the repertoire of CK2 inhibitors to target DYRK and PIMkinases,” Biochim Biophys Acta 1834(7):1402-1409 (2013);triazolo[4,5-b]pyridines as described in Saluste et al.,“Fragment-hopping-based discovery of a novel chemical series ofproto-oncogene PIM-1 kinase inhibitors,” PLoS One 7(10:e45964 (2012);PJ34 as described in Antolin et al., “Identification of pim kinases asnoel targets for PJ34 with confounding effects in PARP biology,” ACSChem Biol. 7(12):1962-1967 (2012); as described in Ogawa et al.,“Insights from Pim1 structure for anti-cancer drug design,” Expert OpinDrug Discov. 7(12):1177-1192 (2012); as described in Brault et al., “PIMkinases are progression markers and emerging therapeutic targets indiffuse large B-cell lymphoma,” Br J Cancer 107(3):491-500 (2012); asdescribed in Nakano et al., “Rational evolution of a novel type ofpotent and selective proviral integration site in Moloney murineleukemia virus kinase 1 (PIM1) inhibitor from a screening-hit compound,”55(11):5151-5164 (2012); as described in Hill et al., “Targeting diversesignaling interaction sites allows the rapid generation of bivalentkinase inhibitors,” ACS Chem Biol 7(3):487-495 (2012); as described inHuber et al., “7,8-dichloro-1-oxo-β-carbolines as a versatile scaffoldfor the development of potent and selective kinase inhibitors withunusual binding modes,” J Med Chem 55(1):403-413 (2012); as described inMorishita et al., “Cell-permeable carboxyl-terminal p27(Kip1) peptideexhibits anti-tumor activity by inhibiting Pim-1 kinase,” J Biol Chem286(4):2681-2688 (2011); Bullock et al., “Structural basis of inhibitorspecificity of the human protooncogene proviral insertion site inmoloney murine leukemia virus (PIM-1) kinase,” J. Med. Chem.48:7604-7614 (2005); Debreczeni et al., “Ruthenium half-sandwichcomplexes bound to protein kinase Pim-1,” Angew. Chem. Int. Ed. Engl.45:1580-1585 (2006); Bregman et al., “Ruthenium half-sandwich complexesas protein kinase inhibitors: an N-succinimidyl ester for rapidderivatizations of the cyclopentadienyl moiety,” Org. Lett. 8:5465-5468(2006); Pogacic et al., “Structural analysis identifies imidazo[1,2-b]pyridazines as PIM kinase inhibitors with in vitro antileukemicactivity,” Cancer Res. 67:6916-6924 (2007); Cheney et al.,“Identification and structure-activity relationships of substitutedpyridones as inhibitors of Pim-1 kinase,” Bioorg. Med. Chem. Lett.17:1679-1683 (2007); Holder et al., “Comparative molecular fieldanalysis of flavonoid inhibitors of the PIM-1 kinase,” Bioorg. Med.Chem. 15:6463-6473 (2007); Pierce et al., “Docking study yields fournovel inhibitors of the protooncogene Pim-1 kinase,” J. Med. Chem.51:1972-1975 (2008); Tong et al.,“Isoxazolo[3,4-b]quinoline-3,4(1H,9H)-diones as unique, potent andselective inhibitors for Pim-1 and Pim-2 kinases: chemistry, biologicalactivities, and molecular modeling,” Bioorg. Med. Chem. Lett.18:5206-5208 (2008); Xia et al., “Synthesis and evaluation of novelinhibitors of Pim-1 and Pim-2 protein kinases,” J. Med. Chem. 52:74-86(2009); Qian et al, “Hit to lead account of the discovery of a new classof inhibitors of Pim kinases and crystallographic studies revealing anunusual kinase binding mode,” J. Med. Chem. 52:1814-1827 (2009); Tao etal., “Discovery of 3H-benzo[4,5]thieno[3,2-d] pyrimidin-4-ones aspotent, highly selective, and orally bioavailable inhibitors of thehuman protooncogene proviral insertion site in moloney murine leukemiavirus (PIM) kinases,” J. Med. Chem. 52:6621-6636 (2009); Tong et al.,“Isoxazolo[3,4-b]quinoline-3,4(1H,9H)-diones as unique, potent andselective inhibitors for Pim-1 and Pim-2 kinases: chemistry, biologicalactivities, and molecular modeling,” Bioorg med Chem Lett.18(19):5206-5208 (2008); and Pogacic et al., “Structural analysisidentifies imidazo[1,2-b]pyridazines as PIM kinase inhibitors with invitro antileukemic activity,” Cancer Res 67(14):6916-6924 (2007).

In some embodiments, PIM1 inhibitors are described in: U.S. Pat. No.8,889,704; U.S. Pat. No. 8,822,497; U.S. Pat. No. 8,604,217; U.S. Pat.No. 8,557,809; U.S. Pat. No. 8,575,145; U.S. Pat. No. 8,541,576; U.S.Pat. No. 8,435,976; U.S. Pat. No. 8,242,129; U.S. Pat. No. 8,124,649;U.S. Pat. No. 8,138,181; U.S. Pat. No. 8,829,193; U.S. Pat. No.8,710,057; U.S. Pat. No. 8,053,454; U.S. Pat. No. 7,268,136;US2014045835; US20140162999; US20140162998; US20110263664; US2011237600;US2011294789; US2010144751; WO2014048939; WO2014033630; WO2014022752;WO2014170403; WO2013175388; WO2013130660; WO2013066684; WO2013013188;WO2013004984; WO2013005041; WO2012156756; WO2012145617; WO2012129338;WO2012148775; WO2012120415; WO2012225062; WO2012098387; WO2012078777;WO2012020215; WO2011101644; WO2011080510; WO2011079274; WO2011035022;WO2011035019; WO2011031979; WO2011025859; WO2011057784; WO2010135571;and WO2009064486.

In some embodiments, disclosed herein are PIM1 inhibitors such asmitoxantrone, SGI-1776, AZD1208, AZD1897, LGH447, JP_11646, Pim1inhibitor 2, SKI-O-068, CX-6258, AR460770, AR00459339 (Array BiopharmaInc.), miR-33a, Pim-1 inhibitory p27 (Kip1) peptide, LY333′531, K00135,quercetagein (3,3′,4′,5,6,7-hydroxyflavone), or LY294002 in combinationwith a BTK inhibitor for the treatment of a hematological malignancy. Insome embodiments, the the Btk inhibitor is ibrutinib, PCI-45292,PCI-45466, AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation),AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation), AVL-292/CC-292(Avila Therapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited),LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences), ACP-196 (AcertaPharma) or JTE-051 (Japan Tobacco Inc). In some embodiments, the BTKinhibitor is ibrutinib.

In some embodiments, disclosed herein are PIM1 inhibitors such asmitoxantrone, SGI-1776, AZD1208, AZD1897, LGH447, JP_11646, Pim1inhibitor 2, SKI-O-068, CX-6258, AR460770, AR00459339 (Array BiopharmaInc.), miR-33a, Pim-1 inhibitory p27 (Kip1) peptide, LY333′531, K00135,quercetagein (3,3′,4′,5,6,7-hydroxyflavone), or LY294002 in combinationwith ibrutinib for the treatment of a hematological malignancy. In someembodiments, the hematological malignancy is MCL. In some embodiments,the MCL is a primary resistant MCL.

Diagnostic and Therapeutic Methods Biomarkers

Disclosed herein are methods of using biomarkers for stratification ofpatients, for monitoring the progression of a treatment, or foroptimization of a therapeutic regimen. In some embodiments, thebiomarkers are evaluated based on the presence or absence ofmodifications or mutations in the biomarkers, or by expression level. Insome embodiments, the biomarkers include MCL1, IDH1, MALT1, or JAK3. Insome embodiments, the biomarkers include MCL1, IDH1, and MALT1. In someembodiments, the biomarkers include PIM1, PIM2, and/or PIM3.

In some embodiments, disclosed are methods of selecting an individualhaving a hematological malignancy for treatment with a combinationcomprising a TEC inhibitor and an anticancer agent, or monitoring thedisease progression of an individual based on the expression level of atleast one biomarker selected from MALT1 or MCL-1. In some embodiments,the TEC inhibitor is an ITK inhibitor or a BTK inhibitor. In someembodiments, disclosed are methods of selecting an individual having ahematological malignancy for treatment with a combination comprising anITK inhibitor and an anticancer agent, or monitoring the diseaseprogression of an individual based on the expression level of at leastone biomarker selected from MALT1 or MCL-1. In some embodiments,disclosed are methods of selecting an individual having a hematologicalmalignancy for treatment with a combination comprising a BTK inhibitorand an anticancer agent, or monitoring the disease progression of anindividual based on the expression level of at least one biomarkerselected from MALT1 or MCL-1. In some embodiments, the BTK inhibitor isselected from among ibrutinib (PCI-32765), PCI-45292, PCI-45466,AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation), AVL-263/CC-263(Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited),LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences), ACP-196 (AcertaPharma) and JTE-051 (Japan Tobacco Inc). In some embodiments, the BTKinhibitor is ibrutinib. In some embodiments, disclosed are methods ofselecting an individual having a hematological malignancy for treatmentwith a combination comprising ibrutinib and an anticancer agent, ormonitoring the disease progression of an individual based on theexpression level of at least one biomarker selected from MALT1 or MCL-1.In some embodiments, the MCL-1 inhbitor is selected from BI97C10,BI112D1, gossypol, obatoclax, MG-132, MIM1, sabutoclax, and TW-37. Insome embodiments, the MALT1 inhibitor is selected from MI-2, mepazine,thioridazine, and promazine. In some embodiments, MALT1 containscytogenetic abnormalities such as t(11;18)(q21;q21) and/ort(14;18)(q32;q21).

In some embodiments, also disclosed are methods of selecting anindividual having a hematological malignancy for treatment with acombination comprising a TEC inhibitor and an anticancer agent, ormonitoring the disease progression of an individual based on theexpression level of at least one biomarker selected from MALT1 or MCL-1,and one or more additional biomarkers. In some embodiments, the TECinhibitor is an ITK inhibitor or a BTK inhibitor. In some embodiments,disclosed are methods of selecting an individual having a hematologicalmalignancy for treatment with a combination comprising an ITK inhibitorand an anticancer agent, or monitoring the disease progression of anindividual based on the expression level of at least one biomarkerselected from MALT1 or MCL-1, and one or more additional biomarkers. Insome embodiments, disclosed are methods of selecting an individualhaving a hematological malignancy for treatment with a combinationcomprising a BTK inhibitor and an anticancer agent, or monitoring thedisease progression of an individual based on the expression level of atleast one biomarker selected from MALT1 or MCL-1, and one or moreadditional biomarkers. In some embodiments, the BTK inhibitor isselected from among ibrutinib (PCI-32765), PCI-45292, PCI-45466,AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation), AVL-263/CC-263(Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited),LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences), ACP-196 (AcertaPharma) and JTE-051 (Japan Tobacco Inc). In some embodiments, the BTKinhibitor is ibrutinib. In some embodiments, disclosed are methods ofselecting an individual having a hematological malignancy for treatmentwith a combination comprising ibrutinib and an anticancer agent, ormonitoring the disease progression of an individual based on theexpression level of at least one biomarker selected from MALT1 or MCL-1,and one or more additional biomarkers. In some embodiments, the one ormore additional biomarkers include CCL3, CCL4, miR155, or a combinationthereof. In some embodiments, disclosed are methods of selecting anindividual having a hematological malignancy for treatment with acombination comprising ibrutinib and an anticancer agent, or monitoringthe disease progression of an individual based on the expression levelof MALT1, and an additional biomarker. In some embodiments, theadditional biomarker is CARD11. In some embodiments, the additionalbiomarker is a CARD11 containing a mutation. In some embodiments, themutation is at amino acid residue position 225, according to thesequence as shown in Table 40. In some embodiments, the mutation isL225LI. In some embodiments, the MCL-1 inhbitor is selected fromBI97C10, BI112D1, gossypol, obatoclax, MG-132, MIM1, sabutoclax, andTW-37. In some embodiments, the MALT1 inhibitor is selected from MI-2,mepazine, thioridazine, and promazine.

In some embodiments, the expression levels of MALT1 and MCL-1 are0.5-fold, 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold,4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold,8.5-fold, 9-fold, 9.5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 75-fold,100-fold, 200-fold, 500-fold, 1000-fold, or more compared to thereference levels of MALT1 and MCL-1. In some embodiments, the expressionlevels of MALT1 and MCL-1 are 0.5-fold, 1-fold, 1.5-fold, 2-fold,2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold,6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold,15-fold, 20-fold, 50-fold, 75-fold, 100-fold, 200-fold, 500-fold,1000-fold, or less compared to the reference levels of MALT1 and MCL-1.

In some embodiments, the reference level is the expression level ofMALT1 and MCL-1 in an individual who does not have a hematologicalmalignancy. In some embodiments, the reference level is the expressionlevel of MALT1 and MCL-1 in an individual prior to treatment with acombination of a TEC inhibitor and an inhibitor of MALT1 or MCL-1.

In some embodiments, disclosed are methods of selecting an individualhaving a hematological malignancy for treatment with a combinationcomprising a TEC inhibitor and an anticancer agent, monitoring thedisease progression of an individual, or optimize the therapeuticregimen in an individual, based on the presence or absence of mutationsin IDH1. In some embodiments, the TEC inhibitor is an ITK inhibitor or aBTK inhibitor. In some embodiments, disclosed are methods of selectingan individual having a hematological malignancy for treatment with acombination comprising an ITK inhibitor and an anticancer agent,monitoring the disease progression of an individual, or optimize thetherapeutic regimen in an individual, based on the presence or absenceof mutations in IDH1. In some embodiments, disclosed are methods ofselecting an individual having a hematological malignancy for treatmentwith a combination comprising a BTK inhibitor and an anticancer agent,monitoring the disease progression of an individual, or optimize thetherapeutic regimen in an individual, based on the presence or absenceof mutations in IDH1. In some embodiments, the BTK inhibitor is selectedfrom among ibrutinib (PCI-32765), PCI-45292, PCI-45466, AVL-101/CC-101(Avila Therapeutics/Celgene Corporation), AVL-263/CC-263 (AvilaTherapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited),LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences), ACP-196 (AcertaPharma) and JTE-051 (Japan Tobacco Inc). In some embodiments, the BTKinhibitor is ibrutinib. In some embodiments, disclosed are methods ofselecting an individual having a hematological malignancy for treatmentwith a combination comprising ibrutinib and an anticancer agent,monitoring the disease progression of an individual, or optimize thetherapeutic regimen in an individual, based on the presence or absenceof mutations in IDH1. In some embodiments, the mutations occur atarginine at amino acid position 132 and/or at arginine at amino acidposition 100 in IDH1. In some embodiments, the mutation occurs atarginine at amino acid position 132 in IDH1. In some embodiments,arginine at amino acid position 132 is converted to an amino acidselected from histidine, serine, cysteine, glycine, or leucine. In someembodiments, arginine at amino acid position 132 is converted tohistidine. In some embodiments, the IDH1 inhibitor is selected fromAGI-5198, AG-120, IDH-C227, and ML309.

In some embodiments, disclosed are methods of selecting an individualhaving a hematological malignancy for treatment with a combinationcomprising a TEC inhibitor and an anticancer agent, monitoring thedisease progression of an individual, or optimize the therapeuticregimen in an individual, based on the presence or absence of mutationsin IDH1, and one or more additional biomarkers. In some embodiments, theTEC inhibitor is an ITK inhibitor or a BTK inhibitor. In someembodiments, disclosed are methods of selecting an individual having ahematological malignancy for treatment with a combination comprising anITK inhibitor and an anticancer agent, monitoring the diseaseprogression of an individual, or optimize the therapeutic regimen in anindividual, based on the presence or absence of mutations in IDH1, andone or more additional biomarkers. In some embodiments, disclosed aremethods of selecting an individual having a hematological malignancy fortreatment with a combination comprising a BTK inhibitor and ananticancer agent, monitoring the disease progression of an individual,or optimize the therapeutic regimen in an individual, based on thepresence or absence of mutations in IDH1, and one or more additionalbiomarkers. In some embodiments, the BTK inhibitor is selected fromamong ibrutinib (PCI-32765), PCI-45292, PCI-45466, AVL-101/CC-101 (AvilaTherapeutics/Celgene Corporation), AVL-263/CC-263 (AvilaTherapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited),LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences), ACP-196 (AcertaPharma) and JTE-051 (Japan Tobacco Inc). In some embodiments, the BTKinhibitor is ibrutinib. In some embodiments, disclosed are methods ofselecting an individual having a hematological malignancy for treatmentwith a combination comprising ibrutinib and an anticancer agent,monitoring the disease progression of an individual, or optimize thetherapeutic regimen in an individual, based on the presence or absenceof mutations in IDH1, and one or more additional biomarkers. In someembodiments, the mutations occur at arginine at amino acid position 132and/or at arginine at amino acid position 100 in IDH1. In someembodiments, the mutation occurs at arginine at amino acid position 132in IDH1. In some embodiments, arginine at amino acid position 132 isconverted to an amino acid selected from histidine, serine, cysteine,glycine, or leucine. In some embodiments, arginine at amino acidposition 132 is converted to histidine. In some embodiments, the IDH1inhibitor is selected from AGI-5198, AG-120, IDH-C227, and ML309.

In some embodiment, the one or more additional biomarkers include amutation or modification in BTK. In some embodiments, the modificationis a mutation at amino acid position 481 in BTK. In some embodiments,the mutation is C481S in BTK. In some embodiments, the C481 mutation inBTK is accompanied with additional mutations in BTK. In someembodiments, the additional mutations in BTK include substitutions atamino acid positions L11, K12, S14, K19, F25, K27, R28, R33, Y39, Y40,E41, 161, V64, R82, Q103, V113, S115, T117, Q127, C154, C155, T184,P189, P190, Y223, W251, R288, L295, G302, R307, D308, V319, Y334, L358,Y361, H362, H364, N365, S366, L369, 1370M, R372, L408, G414, Y418, 1429,K430, E445, G462, Y476, M477, C502, C506, A508, M509, L512, L518, R520,D521, A523, R525, N526, V535, L542, R544, Y551, F559, R562, W563, E567,S578, W581, A582, F583, M587, E589, S592, G594, Y598, A607, G613, Y617,P619, A622, V626, M630, C633, R641, F644, L647, L652, V1065, and A1185.In some embodiments, the additional modifications is selected from amongL11P, K12R, S14F, K19E, F25S, K27R, R28H, R28C, R28P, T33P, Y3S9, Y40C,Y40N, E41K, I61N, V64F, V64D, R82K, Q103QSFSSVR, V113D, S115F, T117P,Q127H, C154S, C155G, T184P, P189A, Y223F, W251L, R288W, R288Q, L295P,G302E, R307K, R307G, R307T, D308E, V319A, Y334S, L358F, Y361C, H362Q,H364P, N365Y, S366F, L369F, 1370M, R372G, L408P, G414R, Y418H, I429N,K430E, E445D, G462D, G462V, Y476D, M477R, C502F, C502W, C506Y, C506R,A508D, M5091, M509V, L512P, L512Q, L518R, R520Q, D521G, D521H, D521N,A523E, R525G, R525P, R525Q, N526K, V535F, L542P, R544G, R544K, Y551F,F559S, R562W, R562P, W563L, E567K, S578Y, W581R, A582V, F583S, M587L,E589D, E589K, E589G, S592P, G594E, Y598C, A607D, G613D, Y617E, P619A,P619S, A622P, V626G, M6301, M630K, M630T, C633Y, R641C, F644L, F644S,L647P, L652P, V10651, and A1185V.

In some embodiments, the one or more additional biomarkers include amutation in PLCγ2. In some embodiments, the mutation in PLCγ2 is amutation at amino acid residue 665, 707, or a combination thereof. Insome embodiments, the mutation is R665W and S707F.

In some embodiments, the one or more additional biomarkers includecytogenetic abnormalities such as del(17p13.1), del(13q14.3),del(11q22.3), del(11q23), unmutated IgVH together with ZAP-70+ and/orCD38+, p53, trisomy 12, t(11;14)(q13;q32), t(14;19)(q32;q13),t(2;14)(p13;q32), del(13q14), +(12q21), del(6q21), ATM del, p53 del,t(15;17); t(8;21)(q22;q22), t(6;9), inv(16)(p13q22), del(16q); inv(16),t(16;16), del(11q), t(9;11), t(11;19), t(1;22), del(5q), +8, +21, +22,del(7q), del(9q), abnormal 11q23, −5, −7, abnormal 3q, complexkaryotype, t(14;19), t(3:14), t(11;14), t(2,8)(p11;q24),t(1;8)(p36;q24), t(8:9)(q24;p13), t(9;14)(p13;q32), t(3:14)(q27;q32), ora combination thereof.

In some embodiments, disclosed are methods of selecting an individualhaving a hematological malignancy for treatment with a combinationcomprising a TEC inhibitor and an anticancer agent, monitoring thedisease progression of an individual, or optimize the therapeuticregimen in an individual, based on the presence or absence of mutationsin IDH1, and a mutation in BTK at amino acid residue position 481. Insome embodiments, the TEC inhibitor is an ITK inhibitor or a BTKinhibitor. In some embodiments, disclosed are methods of selecting anindividual having a hematological malignancy for treatment with acombination comprising an ITK inhibitor and an anticancer agent,monitoring the disease progression of an individual, or optimize thetherapeutic regimen in an individual, based on the presence or absenceof mutations in IDH1, and a mutation in BTK at amino acid residueposition 481. In some embodiments, disclosed are methods of selecting anindividual having a hematological malignancy for treatment with acombination comprising a BTK inhibitor and an anticancer agent,monitoring the disease progression of an individual, or optimize thetherapeutic regimen in an individual, based on the presence or absenceof mutations in IDH1, and a mutation in BTK at amino acid residueposition 481. In some embodiments, the BTK inhibitor is selected fromamong ibrutinib (PCI-32765), PCI-45292, PCI-45466, AVL-101/CC-101 (AvilaTherapeutics/Celgene Corporation), AVL-263/CC-263 (AvilaTherapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited),LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences), ACP-196 (AcertaPharma) and JTE-051 (Japan Tobacco Inc). In some embodiments, the BTKinhibitor is ibrutinib. In some embodiments, disclosed are methods ofselecting an individual having a hematological malignancy for treatmentwith a combination comprising ibrutinib and an anticancer agent,monitoring the disease progression of an individual, or optimize thetherapeutic regimen in an individual, based on the presence or absenceof mutations in IDH1, and a mutation in BTK at amino acid residueposition 481. In some embodiments, the mutations occur at arginine atamino acid position 132 and/or at arginine at amino acid position 100 inIDH1. In some embodiments, the mutation occurs at arginine at amino acidposition 132 in IDH1. In some embodiments, arginine at amino acidposition 132 is converted to an amino acid selected from histidine,serine, cysteine, glycine, or leucine. In some embodiments, arginine atamino acid position 132 is converted to histidine. In some embodiments,the IDH1 inhibitor is selected from AGI-5198, AG-120, IDH-C227, andML309.

In some embodiments, disclosed are methods of selecting an individualhaving a hematological malignancy for treatment with a combinationcomprising a TEC inhibitor and an anticancer agent, monitoring thedisease progression of an individual, or optimize the therapeuticregimen in an individual, based on the presence or absence of mutationsin IDH1, and a mutation in PLCγ2 at amino acid residue position 665and/or 707. In some embodiments, the TEC inhibitor is an ITK inhibitoror a BTK inhibitor. In some embodiments, disclosed are methods ofselecting an individual having a hematological malignancy for treatmentwith a combination comprising an ITK inhibitor and an anticancer agent,monitoring the disease progression of an individual, or optimize thetherapeutic regimen in an individual, based on the presence or absenceof mutations in IDH1, and a mutation in PLCγ2 at amino acid residueposition 665 and/or 707. In some embodiments, disclosed are methods ofselecting an individual having a hematological malignancy for treatmentwith a combination comprising a BTK inhibitor and an anticancer agent,monitoring the disease progression of an individual, or optimize thetherapeutic regimen in an individual, based on the presence or absenceof mutations in IDH1, and a mutation in PLCγ2 at amino acid residueposition 665 and/or 707. In some embodiments, the BTK inhibitor isselected from among ibrutinib (PCI-32765), PCI-45292, PCI-45466,AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation), AVL-263/CC-263(Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited),LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences), ACP-196 (AcertaPharma) and JTE-051 (Japan Tobacco Inc). In some embodiments, the BTKinhibitor is ibrutinib. In some embodiments, disclosed are methods ofselecting an individual having a hematological malignancy for treatmentwith a combination comprising ibrutinib and an anticancer agent,monitoring the disease progression of an individual, or optimize thetherapeutic regimen in an individual, based on the presence or absenceof mutations in IDH1, and a mutation in PLCγ2 at amino acid residueposition 665 and/or 707. In some embodiments, the mutations occur atarginine at amino acid position 132 and/or at arginine at amino acidposition 100 in IDH1. In some embodiments, the mutation occurs atarginine at amino acid position 132 in IDH1. In some embodiments,arginine at amino acid position 132 is converted to an amino acidselected from histidine, serine, cysteine, glycine, or leucine. In someembodiments, arginine at amino acid position 132 is converted tohistidine. In some embodiments, the IDH1 inhibitor is selected fromAGI-5198, AG-120, IDH-C227, and ML309.

In some embodiments, disclosed are methods of selecting an individualhaving a hematological malignancy for treatment with a combinationcomprising a TEC inhibitor and an anticancer agent, monitoring thedisease progression of an individual, or optimize the therapeuticregimen in an individual, based on the presence or absence of mutationsin IDH1, and one or more cytogenetic abnormalities. In some embodiments,the TEC inhibitor is an ITK inhibitor or a BTK inhibitor. In someembodiments, disclosed are methods of selecting an individual having ahematological malignancy for treatment with a combination comprising anITK inhibitor and an anticancer agent, monitoring the diseaseprogression of an individual, or optimize the therapeutic regimen in anindividual, based on the presence or absence of mutations in IDH1, andone or more cytogenetic abnormalities. In some embodiments, disclosedare methods of selecting an individual having a hematological malignancyfor treatment with a combination comprising a BTK inhibitor and ananticancer agent, monitoring the disease progression of an individual,or optimize the therapeutic regimen in an individual, based on thepresence or absence of mutations in IDH1, and one or more cytogeneticabnormalities. In some embodiments, the BTK inhibitor is selected fromamong ibrutinib (PCI-32765), PCI-45292, PCI-45466, AVL-101/CC-101 (AvilaTherapeutics/Celgene Corporation), AVL-263/CC-263 (AvilaTherapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited),LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences), ACP-196 (AcertaPharma) and JTE-051 (Japan Tobacco Inc). In some embodiments, the BTKinhibitor is ibrutinib. In some embodiments, disclosed are methods ofselecting an individual having a hematological malignancy for treatmentwith a combination comprising ibrutinib and an anticancer agent,monitoring the disease progression of an individual, or optimize thetherapeutic regimen in an individual, based on the presence or absenceof mutations in IDH1, and one or more cytogenetic abnormalities. In someembodiments, the mutations occur at arginine at amino acid position 132and/or at arginine at amino acid position 100 in IDH1. In someembodiments, the mutation occurs at arginine at amino acid position 132in IDH1. In some embodiments, arginine at amino acid position 132 isconverted to an amino acid selected from histidine, serine, cysteine,glycine, or leucine. In some embodiments, arginine at amino acidposition 132 is converted to histidine. In some embodiments, the IDH1inhibitor is selected from AGI-5198, AG-120, IDH-C227, and ML309. Insome embodiments, the one or more additional biomarkers includecytogenetic abnormalities such as del(17p13.1), del(13q14.3),del(11q22.3), del(11q23), unmutated IgVH together with ZAP-70+ and/orCD38+, p53, trisomy 12, t(11;14)(q13;q32), t(14;19)(q32;q13),t(2;14)(p13;q32), del(13q14), +(12q21), del(6q21), ATM del, p53 del,t(15;17); t(8;21)(q22;q22), t(6;9), inv(16)(p13q22), del(16q); inv(16),t(16;16), del(11q), t(9;11), t(11;19), t(1;22), del(5q), +8, +21, +22,del(7q), del(9q), abnormal 11q23, −5, −7, abnormal 3q, complexkaryotype, t(14;19), t(3:14), t(11;14), t(2,8)(p11;q24),t(1;8)(p36;q24), t(8:9)(q24;p13), t(9;14)(p13;q32), t(3:14)(q27;q32), ora combination thereof.

In some embodiments, a method of selecting an individual having ahematological malignancy for therapy with a combination comprising a BTKinhibitor and a PIM inhibitor is provided. In some embodiments, the PIMinhibitor is a PIM1 inhibitor. In some embodiments, the PIM inhibitor isa pan-PIM inhibitor. The method may include the step of measuring anexpression level of PIM1 in a sample from the individual; comparing theexpression level of PIM1 with a reference level, and characterizing theindividual as a candidate for therapy with the combination comprising aBTK inhibitor and a PIM inhibitor if the individual has an elevatedlevel of PIM1 compared to the reference level. In some embodiments, themethod may include the step of measuring an expression level of PIM1,PIM2, and/or PIM3 in a sample from the individual; comparing theexpression level of PIM1, PIM2, and/or PIM3 with a reference level, andcharacterizing the individual as a candidate for therapy with thecombination comprising a BTK inhibitor and a PIM inhibitor if theindividual has an elevated level of PIM1, PIM2, and/or PIM3 compared tothe reference level. In some embodiments, the elevated level of PIM1 is1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold,40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold,80-fold, 85-fold, 90-fold, 95-fold, 100-fold, or higher compared to theexpression of the reference level. In some embodiments, thehematological malignancy is a B-cell malignancy. In some embodiments,the B-cell malignancy is an ibrutinib-resistant B-cell malignancy. Insome embodiments, the hematological malignancy is a T-cell malignancy.In some embodiments, the reference level is the expression level of PIM1in an individual that does not have a B-cell malignancy. In someembodiments, the reference level is the expression level of PIM1 in anindividual prior to treatment with a combination of a BTK inhibitor anda PIM inhibitor. In some embodiments, the reference level is theexpression level of PIM1 in an individual after treatment with a BTKinhibitor.

In some embodiments, a method of selecting an individual having ahematological malignancy for therapy with a combination comprising a BTKinhibitor and a PIM inhibitor is provided. In some embodiments, the PIMinhibitor is a PIM1 inhbitor. In some embodiments, the PIM inhibitor isa pan-PIM inhibitor. The method may include the step of measuring anexpression level of PIM1, PIM2, and/or PIM3 in a sample from theindividual; comparing the expression level of PIM1, PIM2, and/or PIM3with a reference level for PIM1, PIM2, and/or PIM3, and characterizingthe individual as a candidate for therapy with the combinationcomprising a BTK inhibitor and a PIM inhibitor if the individual has anelevated level of PIM1, PIM2, and/or PIM3 compared to the referencelevel of PIM1, PIM2, and/or PIM3. In some embodiments, the elevatedlevel of PIM1, PIM2, or PIM3 is 1-fold, 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold,20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold,60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold,100-fold, or higher compared to the expression of the reference level.In some embodiments, the hematological malignancy is a B-cellmalignancy. In some embodiments, the B-cell malignancy is anibrutinib-resistant B-cell malignancy. In some embodiments, thereference level is the expression level of PIM1, PIM2, and/or PIM3 is inan individual that does not have a B-cell malignancy. In someembodiments, the reference level is the expression level of PIM1, PIM2,and/or PIM3 in an individual prior to treatment with a combination of aBTK inhibitor and a PIM inhibitor.

In some embodiments, a method of assessing whether a subject having ahematological malignancy is less responsive or likely to be lessresponsive to therapy with a BTK inhibitor is provided. The method mayinclude the steps of testing a sample containing a nucleic acid moleculeencoding a PIM1 polypeptide from the subject. In some embodiments, themethod may include the step of determining whether the encoded PIM1polypeptide is modified at certain positions, such as at amino acidresidue position 2, 81, and/or 97 of the amino acid sequence as setforth in SEQ. ID NO:1; and characterizing the subject as resistant orlikely to become resistant to therapy with a BTK inhibitor if thesubject has the modification at amino acid position 2, 81, and/or 97.The modification may comprise a substitution, an addition, or a deletionof the amino acid at amino acid position 2, 81, or 97 in the PIM1polypeptide. Exemplary modifications include: PIM1 L2V; PIM1 P81S; orPIM1 S97N. Additional modifications of PIM1 as set forth in SEQ ID NO.1, the presence of which may characterize the subject as resistant orlikely to become resistant to therapy with a BTK inhibitor include: M11;L2F; P16S; C17S; G28D/G28V; K29N/K29fs*18; E30K; E32K; P33fs*68; Q127;Q37H; G55D; 166M; H68D; E70Q; P81A; P87T; E89K; V90L/V90fs*27; L93V;S97T; P125S; V126M; Q127*; E135Q; E142fs*132; H165Y; E171K; L174F;1175V; E181D; L184F; and/or L193F. In some embodiments, thehematological malignancy is a B-cell malignancy.

In some embodiments, a method of selecting an individual having ahematological malignancy for treatment with a combination comprising aBTK inhibitor and a PIM inhibitor is provided. In some embodiments, thePIM inhibitor is a PIM1 inhibitor. In some embodiments, the PIMinhibitor is a pan-PIM inhibitor. The method may include the step ofmonitoring the disease progression of an individual and/or optimizingthe therapeutic regimen of the individual, based on the presence orabsence of modifications in PIM1, PIM2, and/or PIM3. Exemplarymodifications include, but or not limited to, substitutions, additions,or deletions at amino acid position 2, 81, and/or 97 of the PIM1polypeptide as set forth in SEQ. ID NO. 1. In some embodiments, thehematological malignancy is a B-cell malignancy.

Diagnostic Methods

Methods for determining the expression or presence of biomarkers such asMALT1, MCL1, IDH1, JAK3, PIM1, PIM2, and PIM3 are well known in the art.Circulating levels of biomarkers in a blood sample obtained from acandidate subject are measured, for example, by ELISA, radioimmunoassay(RIA), electrochemiluminescence (ECL), Western blot, multiplexingtechnologies, or other similar methods. Cell surface expression ofbiomarkers are measured, for example, by flow cytometry,immunohistochemistry, Western Blot, immunoprecipitation, magnetic beadselection, and quantification of cells expressing either of these cellsurface markers. Biomarker RNA expression levels could be measured byRT-PCR, Qt-PCR, microarray, Northern blot, or other similartechnologies.

As disclosed herein, determining the expression or presence of thebiomarker of interest at the protein or nucleotide level areaccomplished using any detection method known to those of skill in theart. By “detecting expression” or “detecting the level of is intendeddetermining the expression level or presence of a biomarker protein orgene in the biological sample. Thus, “detecting expression” encompassesinstances where a biomarker is determined not to be expressed, not to bedetectably expressed, expressed at a low level, expressed at a normallevel, or overexpressed.

In certain aspects of the method provided herein, the one or moresubpopulation of lymphocytes are isolated, detected or measured. Incertain embodiments, the one or more subpopulation of lymphocytes areisolated, detected or measured using immunophenotyping techniques. Inother embodiments, the one or more subpopulation of lymphocytes areisolated, detected or measured using fluorescence activated cell sorting(FACS) techniques.

In certain aspects, the expression or presence of these variousbiomarkers and any clinically useful prognostic markers in a biologicalsample are detected at the protein or nucleic acid level, using, forexample, immunohistochemistry techniques or nucleic acid-basedtechniques such as in situ hybridization and RT-PCR. In one embodiments,the expression or presence of one or more biomarkers is carried out by ameans for nucleic acid amplification, a means for nucleic acidsequencing, a means utilizing a nucleic acid microarray (DNA and RNA),or a means for in situ hybridization using specifically labeled probes.

In other embodiments, the determining the expression or presence of oneor more biomarkers is carried out through gel electrophoresis. In oneembodiment, the determination is carried out through transfer to amembrane and hybridization with a specific probe.

In other embodiments, the determining the expression or presence of oneor more biomarkers carried out by a diagnostic imaging technique.

In still other embodiments, the determining the expression or presenceof one or more biomarkers carried out by a detectable solid substrate.In one embodiment, the detectable solid substrate is paramagneticnanoparticles functionalized with antibodies.

In another aspect, provided herein are methods for detecting ormeasuring residual lymphoma following a course of treatment in order toguide continuing or discontinuing treatment or changing from onetherapeutic regimen to another comprising determining the expression orpresence of one or more biomarkers from one or more subpopulation oflymphocytes in a subject wherein the course of treatment is treatmentwith a Btk inhibitor (e.g., ibrutinib).

Methods for detecting expression of the biomarkers described herein,within the test and control biological samples comprise any methods thatdetermine the quantity or the presence of these markers either at thenucleic acid or protein level. Such methods are well known in the artand include but are not limited to western blots, northern blots, ELISA,immunoprecipitation, immunofluorescence, flow cytometry,immunohistochemistry, nucleic acid hybridization techniques, nucleicacid reverse transcription methods, and nucleic acid amplificationmethods. In particular embodiments, expression of a biomarker isdetected on a protein level using, for example, antibodies that aredirected against specific biomarker proteins. These antibodies are usedin various methods such as Western blot, ELISA, multiplexingtechnologies, immunoprecipitation, or immunohistochemistry techniques.In some embodiments, detection of biomarkers is accomplished by ELISA.In some embodiments, detection of biomarkers is accomplished byelectrochemiluminescence (ECL).

Any means for specifically identifying and quantifying a biomarker (forexample, biomarker, a biomarker of cell survival or proliferation, abiomarker of apoptosis, a biomarker of a Btk-mediated signaling pathway)in the biological sample of a candidate subject is contemplated. Thus,in some embodiments, expression level of a biomarker protein of interestin a biological sample is detected by means of a binding protein capableof interacting specifically with that biomarker protein or abiologically active variant thereof. In some embodiments, labeledantibodies, binding portions thereof, or other binding partners areused. The word “label” when used herein refers to a detectable compoundor composition that is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. In some embodiments, the labelis detectable by itself (e.g., radioisotope labels or fluorescentlabels) or, in the case of an enzymatic label, catalyzes chemicalalteration of a substrate compound or composition that is detectable.

The antibodies for detection of a biomarker protein are eithermonoclonal or polyclonal in origin, or are synthetically orrecombinantly produced. The amount of complexed protein, for example,the amount of biomarker protein associated with the binding protein, forexample, an antibody that specifically binds to the biomarker protein,is determined using standard protein detection methodologies known tothose of skill in the art. A detailed review of immunological assaydesign, theory and protocols are found in numerous texts in the art(see, for example, Ausubel et al., eds. (1995) Current Protocols inMolecular Biology) (Greene Publishing and Wiley-Interscience, NY));Coligan et al., eds. (1994) Current Protocols in Immunology (John Wiley& Sons, Inc., New York, N.Y.).

The choice of marker used to label the antibodies will vary dependingupon the application. However, the choice of the marker is readilydeterminable to one skilled in the art. These labeled antibodies areused in immunoassays as well as in histological applications to detectthe presence of any biomarker or protein of interest. The labeledantibodies are either polyclonal or monoclonal. Further, the antibodiesfor use in detecting a protein of interest are labeled with aradioactive atom, an enzyme, a chromophoric or fluorescent moiety, or acolorimetric tag as described elsewhere herein. The choice of tagginglabel also will depend on the detection limitations desired. Enzymeassays (ELISAs) typically allow detection of a colored product formed byinteraction of the enzyme-tagged complex with an enzyme substrate.Radionuclides that serve as detectable labels include, for example,1-131, 1-123, 1-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, andPd-109. Examples of enzymes that serve as detectable labels include, butare not limited to, horseradish peroxidase, alkaline phosphatase,beta-galactosidase, and glucose-6-phosphate dehydrogenase. Chromophoricmoieties include, but are not limited to, fluorescein and rhodamine. Theantibodies are conjugated to these labels by methods known in the art.For example, enzymes and chromophoric molecules are conjugated to theantibodies by means of coupling agents, such as dialdehydes,carbodiimides, dimaleimides, and the like. Alternatively, conjugationoccurs through a ligand-receptor pair. Examples of suitableligand-receptor pairs are biotin-avidin or biotin-streptavidin, andantibody-antigen.

In certain embodiments, expression or presence of one or more biomarkersor other proteins of interest within a biological sample, for example, asample of bodily fluid, is determined by radioimmunoassays orenzyme-linked immunoassays (ELISAs), competitive binding enzyme-linkedimmunoassays, dot blot (see, for example, Promega Protocols andApplications Guide, Promega Corporation (1991), Western blot (see, forexample, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual,Vol. 3, Chapter 18 (Cold Spring Harbor Laboratory Press, Plainview,N.Y.), chromatography such as high performance liquid chromatography(HPLC), or other assays known in the art. Thus, the detection assaysinvolve steps such as, but not limited to, immunoblotting,immunodiffusion, immunoelectrophoresis, or immunoprecipitation.

In certain other embodiments, the methods of the invention are usefulfor identifying and treating cancer, including those listed above, thatare refractory to (i.e., resistant to, or have become resistant to)first-line oncotherapeutic treatments.

In some embodiments, the expression or presence of one or more of thebiomarkers described herein are also determined at the nucleic acidlevel. Nucleic acid-based techniques for assessing expression are wellknown in the art and include, for example, determining the level ofbiomarker mRNA in a biological sample. Many expression detection methodsuse isolated RNA. Any RNA isolation technique that does not selectagainst the isolation of mRNA is utilized for the purification of RNA(see, e.g., Ausubel et al., ed. (1987-1999) Current Protocols inMolecular Biology (John Wiley & Sons, New York). Additionally, largenumbers of tissue samples are readily processed using techniques wellknown to those of skill in the art, such as, for example, thesingle-step RNA isolation process disclosed in U.S. Pat. No. 4,843,155.

Thus, in some embodiments, the detection of a biomarker or other proteinof interest is assayed at the nucleic acid level using nucleic acidprobes. The term “nucleic acid probe” refers to any molecule that iscapable of selectively binding to a specifically intended target nucleicacid molecule, for example, a nucleotide transcript. Probes aresynthesized by one of skill in the art, or derived from appropriatebiological preparations. Probes are specifically designed to be labeled,for example, with a radioactive label, a fluorescent label, an enzyme, achemiluminescent tag, a colorimetric tag, or other labels or tags thatare discussed above or that are known in the art. Examples of moleculesthat are utilized as probes include, but are not limited to, RNA andDNA.

For example, isolated mRNA are used in hybridization or amplificationassays that include, but are not limited to, Southern or Northernanalyses, polymerase chain reaction analyses and probe arrays. Onemethod for the detection of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that hybridize to the mRNAencoded by the gene being detected. The nucleic acid probe comprises of,for example, a full-length cDNA, or a portion thereof, such as anoligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotidesin length and sufficient to specifically hybridize under stringentconditions to an mRNA or genomic DNA encoding a biomarker, biomarkerdescribed herein above. Hybridization of an mRNA with the probeindicates that the biomarker or other target protein of interest isbeing expressed.

In one embodiment, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in a gene chip array. A skilled artisan readilyadapts known mRNA detection methods for use in detecting the level ofmRNA encoding the biomarkers or other proteins of interest.

An alternative method for determining the level of an mRNA of interestin a sample involves the process of nucleic acid amplification, e.g., byRT-PCR (see, for example, U.S. Pat. No. 4,683,202), ligase chainreaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189 193),self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl.Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwohet al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi et al. (1988) Bio/Technology 6:1197), rolling circlereplication (U.S. Pat. No. 5,854,033) or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers. In particular aspects of the invention, biomarker expression isassessed by quantitative fluorogenic RT-PCR (i.e., the TaqMan0 System).

Expression levels of an RNA of interest are monitored using a membraneblot (such as used in hybridization analysis such as Northern, dot, andthe like), or microwells, sample tubes, gels, beads or fibers (or anysolid support comprising bound nucleic acids). See U.S. Pat. Nos.5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which areincorporated herein by reference. The detection of expression alsocomprises using nucleic acid probes in solution.

In one embodiment of the invention, microarrays are used to determineexpression or presence of one or more biomarkers. Microarrays areparticularly well suited for this purpose because of the reproducibilitybetween different experiments. DNA microarrays provide one method forthe simultaneous measurement of the expression levels of large numbersof genes. Each array consists of a reproducible pattern of captureprobes attached to a solid support. Labeled RNA or DNA is hybridized tocomplementary probes on the array and then detected by laser scanningHybridization intensities for each probe on the array are determined andconverted to a quantitative value representing relative gene expressionlevels. See, U.S. Pat. Nos. 6,040,138, 5,800,992 and 6,020,135,6,033,860, and 6,344,316, which are incorporated herein by reference.High-density oligonucleotide arrays are particularly useful fordetermining the gene expression profile for a large number of RNA's in asample.

Techniques for the synthesis of these arrays using mechanical synthesismethods are described in, e.g., U.S. Pat. No. 5,384,261, incorporatedherein by reference in its entirety. In some embodiments, an array isfabricated on a surface of virtually any shape or even a multiplicity ofsurfaces. In some embodiments, an array is a planar array surface. Insome embodiments, arrays include peptides or nucleic acids on beads,gels, polymeric surfaces, fibers such as fiber optics, glass or anyother appropriate substrate, see U.S. Pat. Nos. 5,770,358, 5,789,162,5,708,153, 6,040,193 and 5,800,992, each of which is hereby incorporatedin its entirety for all purposes. In some embodiments, arrays arepackaged in such a manner as to allow for diagnostics or othermanipulation of an all-inclusive device.

Samples

In some embodiments, the sample for use in the methods is obtained fromcells of a hematological malignant cell line. In some embodiments, thesample is obtained from cells of a acute lymphoblastic leukemia (ALL),acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML),acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL),high risk CLL, small lymphocytic lymphoma (SLL), high risk SLL,follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantlecell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma,extranodal marginal zone B cell lymphoma, nodal marginal zone B celllymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma,primary mediastinal B-cell lymphoma (PMBL), immunoblastic large celllymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocyticleukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma,plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B celllymphoma, intravascular large B cell lymphoma, primary effusionlymphoma, or lymphomatoid granulomatosis cell line. In some embodiments,the sample is obtained from cells of a DLBCL cell line.

In some embodiments, the sample is a DLBCL cell or population of DLBCLcells. In some embodiments, the DLBCL cell line is an activatedB-cell-like (ABC)-DLBCL cell line. In some embodiments, the DLBCL cellline is a germinal center B-cell-like (GCB)-DLBCL cell line. In someembodiments, the DLBCL cell line is OCI-Ly1, OCI-Ly2, OCI-Ly3, OCI-Ly4,OCI-Ly6, OCI-Ly7, OCI-Ly10, OCI-Ly18, OCI-Ly19, U2932, DB, HBL-1, RIVA,SUDHL2, or TMD8. In some embodiments, the DLBCL cell line that issensitive to treatment with a BTK inhibitor is TMD8, HBL-1 or OCI-Ly10.In some embodiments, the DLBCL cell line that is resistant to treatmentwith a BTK inhibitor is OCI-Ly3, DB or OCI-Ly19.

In some embodiments, the sample is a MCL cell or population of MCLcells. In some embodiments, the MCL cell line is Jeko (JeKo-1), SP-53,Granta 519, or REC-1. In some embodiments, the MCL cell line that issensitive to treatment with a BTK inhibitor is Jeko (JeKo-1).

In some embodiments, the sample for use in the methods is from anytissue or fluid from a patient. Samples include, but are not limited, towhole blood, dissociated bone marrow, bone marrow aspirate, pleuralfluid, peritoneal fluid, central spinal fluid, abdominal fluid,pancreatic fluid, cerebrospinal fluid, brain fluid, ascites, pericardialfluid, urine, saliva, bronchial lavage, sweat, tears, ear flow, sputum,hydrocele fluid, semen, vaginal flow, milk, amniotic fluid, andsecretions of respiratory, intestinal or genitourinary tract. Inparticular embodiments, the sample is a blood serum sample. Inparticular embodiments, the sample is from a fluid or tissue that ispart of, or associated with, the lymphatic system or circulatory system.In some embodiments, the sample is a blood sample that is a venous,arterial, peripheral, tissue, cord blood sample. In some embodiments,the sample is a blood cell sample containing one or more peripheralblood mononuclear cells (PBMCs). In some embodiments, the samplecontains one or more circulating tumor cells (CTCs). In someembodiments, the sample contains one or more disseminated tumor cells(DTC, e.g., in a bone marrow aspirate sample).

In some embodiments, the samples are obtained from the individual by anysuitable means of obtaining the sample using well-known and routineclinical methods. Procedures for obtaining fluid samples from anindividual are well known. For example, procedures for drawing andprocessing whole blood and lymph are well-known and can be employed toobtain a sample for use in the methods provided. Typically, forcollection of a blood sample, an anti-coagulation agent (e.g., EDTA, orcitrate and heparin or CPD (citrate, phosphate, dextrose) or comparablesubstances) is added to the sample to prevent coagulation of the blood.In some examples, the blood sample is collected in a collection tubethat contains an amount of EDTA to prevent coagulation of the bloodsample.

In some embodiments, the collection of a sample from the individual isperformed at regular intervals, such as, for example, one day, two days,three days, four days, five days, six days, one week, two weeks, weeks,four weeks, one month, two months, three months, four months, fivemonths, six months, one year, daily, weekly, bimonthly, quarterly,biyearly or yearly.

In some embodiments, the collection of a sample is performed at apredetermined time or at regular intervals relative to treatment with acombination of a TEC inhibitor and an anticancer agent. In someembodiments, the TEC inhibitor is a BTK inhibitor, an ITK inhibitor, aTEC inhibitor, a RLK inhibitor, or a BMX inhibitor. In some embodiments,the TEC inhibitor is an ITK inhibitor. In some embodiments, the TECinhibitor is a BTK inhibitor. In some embodiments, the anticancer agentis an inhibitor of MALT1, JAK3, MCL-1 or IDH1. In some embodiments, theanticancer agent is an inhibitor of MALT1, MCL-1 or IDH1.

In some embodiments, the collection of a sample is performed at apredetermined time or at regular intervals relative to treatment with acombination of an ITK inhibitor and an anticancer agent. For example, asample is collected from a patient at a predetermined time or at regularintervals prior to, during, or following treatment or between successivetreatments with a combination of an ITK inhibitor and an anticanceragent. In particular examples, a sample is obtained from a patient priorto administration of a combination of an ITK inhibitor and an anticanceragent, and then again at regular intervals after treatment with thecombination of the ITK inhibitor and the anticancer agent has beenaffected. In some embodiments, the patient is administered a combinationof an ITK inhibitor and an anticancer agent and one or more additionaltherapeutic agents. In some embodiments, the ITK inhibitor is anirreversible ITK inhibitor. In some embodiments, the ITK inhibitor is areversible ITK inhibitor. In some embodiments, the anticancer agent isan inhibitor of MALT1, JAK3, MCL-1, IDH1, PIM1, PIM2, and/or PIM3. Insome embodiments, the anticancer agent is an inhibitor of MALT1. In someembodiments, a MALT1 inhibitor comprises MI-2, mepazine, thioridazine,and promazine. In some embodiments, the anticancer agent is an inhibitorof JAK3. In some embodiments, a JAK3 inhibitor comprises AT9283,BOT-4-one, cercosporamide, JAK3 Inhibitor IV, JAK3 Inhibitor V, JAK3Inhibitor VI, JAK3 Inhibitor VII, JANEX-1, MS-1020, PF-956980,ruxolitinib, TCS21311, TG101209, tofacitinib, VX-509, WHI-P 131, andWHI-P 154. In some embodiments, the anticancer agent is an inhibitor ofMCL-1. In some embodiments, a MCL-1 inhibitor comprises BI97C10,BI112D1, gossypol, obatoclax, MG-132, MIM1, sabutoclax, and TW-37. Insome embodiments, the anticancer agent is an inhibitor of IDH1. In someembodiments, an IDH1 inhibitor comprises AGI-5198, AG-120, IDH-C227, andML309.

In some embodiments, the collection of a sample is performed at apredetermined time or at regular intervals relative to treatment with acombination of a BTK inhibitor and an anticancer agent. For example, asample is collected from a patient at a predetermined time or at regularintervals prior to, during, or following treatment or between successivetreatments with a combination of a BTK inhibitor and an anticanceragent. In particular examples, a sample is obtained from a patient priorto administration of a combination of a BTK inhibitor and an anticanceragent, and then again at regular intervals after treatment with thecombination of the BTK inhibitor and the anticancer agent has beeneffected. In some embodiments, the patient is administered a combinationof a BTK inhibitor and an anticancer agent and one or more additionaltherapeutic agents. In some embodiments, the BTK inhibitor is anirreversible BTK inhibitor. In some embodiments, the BTK inhibitor is areversible BTK inhibitor. In some embodiments, the BTK inhibitor isibrutinib. In some embodiments, the BTK inhibitor is selected from amongibrutinib (PCI-32765), PCI-45292, PCI-45466, AVL-101/CC-101 (AvilaTherapeutics/Celgene Corporation), AVL-263/CC-263 (AvilaTherapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited),LFM-A13, BGB-3111 (Beigene), KBP-7536 (KBP BioSciences), ACP-196 (AcertaPharma) and JTE-051 (Japan Tobacco Inc). In some embodiments, theanticancer agent is an inhibitor of MALT1, JAK3, MCL-1 or IDH1. In someembodiments, the anticancer agent is an inhibitor of MALT1, MCL-1 orIDH1. In some embodiments, the anticancer agent is an inhibitor ofMALT1. In some embodiments, a MALT1 inhibitor comprises MI-2, mepazine,thioridazine, and promazine. In some embodiments, the anticancer agentis an inhibitor of JAK3. In some embodiments, a JAK3 inhibitor comprisesAT9283, BOT-4-one, cercosporamide, JAK3 Inhibitor IV, JAK3 Inhibitor V,JAK3 Inhibitor VI, JAK3 Inhibitor VII, JANEX-1, MS-1020, PF-956980,ruxolitinib, TCS21311, TG101209, tofacitinib, VX-509, WHI-P 131, andWHI-P 154. In some embodiments, the anticancer agent is an inhibitor ofMCL-1. In some embodiments, a MCL-1 inhibitor comprises BI97C10,BI112D1, gossypol, obatoclax, MG-132, MIM1, sabutoclax, and TW-37. Insome embodiments, the anticancer agent is an inhibitor of IDH1. In someembodiments, an IDH1 inhibitor comprises AGI-5198, AG-120, IDH-C227, andML309.

In some embodiments, the collection of a sample is performed at apredetermined time or at regular intervals relative to treatment with acombination of ibrutinib and an anticancer agent. For example, a sampleis collected from a patient at a predetermined time or at regularintervals prior to, during, or following treatment or between successivetreatments with a combination of ibrutinib and an anticancer agent. Inparticular examples, a sample is obtained from a patient prior toadministration of a combination of ibrutinib and an anticancer agent,and then again at regular intervals after treatment with the combinationof ibrutinib and the anticancer agent has been effected. In someembodiments, the patient is administered a combination of ibrutinib andan anticancer agent and one or more additional therapeutic agents. Insome embodiments, the anticancer agent is an inhibitor of MALT1, JAK3,MCL-1 or IDH1. In some embodiments, the anticancer agent is an inhibitorof MALT1, MCL-1 or IDH1. In some embodiments, the anticancer agent is aninhibitor of MALT1. In some embodiments, a MALT1 inhibitor comprisesMI-2, mepazine, thioridazine, and promazine. In some embodiments, theanticancer agent is an inhibitor of JAK3. In some embodiments, a JAK3inhibitor comprises AT9283, BOT-4-one, cercosporamide, JAK3 InhibitorIV, JAK3 Inhibitor V, JAK3 Inhibitor VI, JAK3 Inhibitor VII, JANEX-1,MS-1020, PF-956980, ruxolitinib, TCS21311, TG101209, tofacitinib,VX-509, WHI-P 131, and WHI-P 154. In some embodiments, the anticanceragent is an inhibitor of MCL-1. In some embodiments, a MCL-1 inhibitorcomprises BI97C10, BI112D1, gossypol, obatoclax, MG-132, MIM1,sabutoclax, and TW-37. In some embodiments, the anticancer agent is aninhibitor of IDH1. In some embodiments, an IDH1 inhibitor comprisesAGI-5198, AG-120, IDH-C227, and ML309.

Additional Combination Therapies

In certain embodiments, a TEC inhibitor and an anticancer agent areadministered in combination with an additional therapeutic agent for thetreatment of a hematological malignancy. In some embodiments, the TECinhibitor is a BTK inhibitor, an ITK inhibitor, a TEC inhibitor, a RLKinhibitor, or a BMX inhibitor. In certain embodiments, an ITK inhibitorand an anticancer agent are administered in combination with anadditional therapeutic agent for the treatment of a hematologicalmalignancy. In certain embodiments, a BTK inhibitor (e.g. ibrutinib) andan anticancer agent are administered in combination with an additionaltherapeutic agent for the treatment of a hematological malignancy. Insome embodiments, the anticancer agent is an inhibitor of MALT1, JAK3,MCL-1, or IDH1. In some embodiments, the anticancer agent is a PIMinhibitor. In some embodiments, the anticancer agent is an inhibitor ofMALT1. In some embodiments, the anticancer agent is an inhibitor ofJAK3. In some embodiments, the anticancer agent is an inhibitor ofMCL-1. In some embodiments, the anticancer agent is an inhibitor ofIDH1. In some embodiments, the additional therapeutic agent is selectedfrom an inhibitor of LYN, SYK, JAK1/2, PI3K, PLCy, MAPK, HDAC, NFKB, orMEK. In some embodiments, the additional therapeutic agent comprises anagent selected from: bendamustine, bortezomib, lenalidomide, idelalisib(GS-1101), vorinostat, ofatumumab, everolimus, panobinostat,temsirolimus, romidepsin, vorinostat, fludarabine, cyclophosphamide,mitoxantrone, pentostatine, prednisone, etopside, procarbazine, andthalidomide.

In some embodiments, the additional therapeutic agent is selected from achemotherapeutic agent, a biologic agent, radiation therapy, bone marrowtransplant or surgery. In some embodiments, the chemotherapeutic agentis selected from among chlorambucil, ifosfamide, doxorubicin,mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus,fludarabine, fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab,dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab,bortezomib, pentostatin, endostatin, or a combination thereof.

In some embodiments, the additional therapeutic agent is selected from:Nitrogen Mustards such as for example, bendamustine, chlorambucil,chlormethine, cyclophosphamide, ifosfamide, melphalan, prednimustine,trofosfamide; Alkyl Sulfonates like busulfan, mannosulfan, treosulfan;Ethylene Imines like carboquone, thiotepa, triaziquone; Nitrosoureaslike carmustine, fotemustine, lomustine, nimustine, ranimustine,semustine, streptozocin; Epoxides such as for example, etoglucid; OtherAlkylating Agents such as for example dacarbazine, mitobronitol,pipobroman, temozolomide; Folic Acid Analogues such as for examplemethotrexate, permetrexed, pralatrexate, raltitrexed; Purine Analogssuch as for example cladribine, clofarabine, fludarabine,mercaptopurine, nelarabine, tioguanine; Pyrimidine Analogs such as forexample azacitidine, capecitabine, carmofur, cytarabine, decitabine,fluorouracil, gemcitabine, tegafur; Vinca Alkaloids such as for examplevinblastine, vincristine, vindesine, vinflunine, vinorelbine;Podophyllotoxin Derivatives such as for example etoposide, teniposide;Colchicine derivatives such as for example demecolcine; Taxanes such asfor example docetaxel, paclitaxel, paclitaxel poliglumex; Other PlantAlkaloids and Natural Products such as for example trabectedin;Actinomycines such as for example dactinomycin; Antracyclines such asfor example aclarubicin, daunorubicin, doxorubicin, epirubicin,idarubicin, mitoxantrone, pirarubicin, valrubicin, zorubincin; OtherCytotoxic Antibiotics such as for example bleomycin, ixabepilone,mitomycin, plicamycin; Platinum Compounds such as for examplecarboplatin, cisplatin, oxaliplatin, satraplatin; Methylhydrazines suchas for example procarbazine; Sensitizers such as for exampleaminolevulinic acid, efaproxiral, methyl aminolevulinate, porfimersodium, temoporfin; Protein Kinase Inhibitors such as for exampledasatinib, erlotinib, everolimus, gefitinib, imatinib, lapatinib,nilotinib, pazonanib, sorafenib, sunitinib, temsirolimus; OtherAntineoplastic Agents such as for example alitretinoin, altretamine,amzacrine, anagrelide, arsenic trioxide, asparaginase, bexarotene,bortezomib, celecoxib, denileukin diftitox, estramustine,hydroxycarbamide, irinotecan, lonidamine, masoprocol, miltefosein,mitoguazone, mitotane, oblimersen, pegaspargase, pentostatin,romidepsin, sitimagene ceradenovec, tiazofurine, topotecan, tretinoin,vorinostat; Estrogens such as for example diethylstilbenol,ethinylestradiol, fosfestrol, polyestradiol phosphate; Progestogens suchas for example gestonorone, medroxyprogesterone, megestrol; GonadotropinReleasing Hormone Analogs such as for example buserelin, goserelin,leuprorelin, triptorelin; Anti-Estrogens such as for examplefulvestrant, tamoxifen, toremifene; Anti-Androgens such as for examplebicalutamide, flutamide, nilutamide, Enzyme Inhibitors,aminoglutethimide, anastrozole, exemestane, formestane, letrozole,vorozole; Other Hormone Antagonists such as for example abarelix,degarelix; Immunostimulants such as for example histaminedihydrochloride, mifamurtide, pidotimod, plerixafor, roquinimex,thymopentin; Immunosuppressants such as for example everolimus,gusperimus, leflunomide, mycophenolic acid, sirolimus; CalcineurinInhibitors such as for example ciclosporin, tacrolimus; OtherImmunosuppressants such as for example azathioprine, lenalidomide,methotrexate, thalidomide; and Radiopharmaceuticals such as for example,iobenguane.

In some embodiments, the additional therapeutic agent is selected from:interferons, interleukins, Tumor Necrosis Factors, Growth Factors, orthe like.

In some embodiments, the additional therapeutic agent is selected from:ancestim, filgrastim, lenograstim, molgramostim, pegfilgrastim,sargramostim; Interferons such as for example interferon alfa natural,interferon alfa-2a, interferon alfa-2b, interferon alfacon-1, interferonalfa-n1, interferon beta natural, interferon beta-1a, interferonbeta-1b, interferon gamma, peginterferon alfa-2a, peginterferon alfa-2b;Interleukins such as for example aldesleukin, oprelvekin; OtherImmunostimulants such as for example BCG vaccine, glatiramer acetate,histamine dihydrochloride, immunocyanin, lentinan, melanoma vaccine,mifamurtide, pegademase, pidotimod, plerixafor, poly I:C, poly ICLC,roquinimex, tasonermin, thymopentin; Immunosuppressants such as forexample abatacept, abetimus, alefacept, antilymphocyte immunoglobulin(horse), antithymocyte immunoglobulin (rabbit), eculizumab, efalizumab,everolimus, gusperimus, leflunomide, muromab-CD3, mycophenolic acid,natalizumab, sirolimus; TNF alpha Inhibitors such as for exampleadalimumab, afelimomab, certolizumab pegol, etanercept, golimumab,infliximab; Interleukin Inhibitors such as for example anakinra,basiliximab, canakinumab, daclizumab, mepolizumab, rilonacept,tocilizumab, ustekinumab; Calcineurin Inhibitors such as for exampleciclosporin, tacrolimus; Other Immunosuppressants such as for exampleazathioprine, lenalidomide, methotrexate, thalidomide.

In some embodiments, the additional therapeutic agent is selected from:Adalimumab, Alemtuzumab, Basiliximab, Bevacizumab, Cetuximab,Certolizumab pegol, Daclizumab, Eculizumab, Efalizumab, Gemtuzumab,Ibritumomab tiuxetan, Infliximab, Muromonab-CD3, Natalizumab,Panitumumab, Ranibizumab, Rituximab, Tositumomab, Trastuzumab, or thelike, or a combination thereof.

In some embodiments, the additional therapeutic agent is selected from:Monoclonal Antibodies such as for example alemtuzumab, bevacizumab,catumaxomab, cetuximab, edrecolomab, gemtuzumab, ofatumumab,panitumumab, rituximab, trastuzumab; Immunosuppressants, eculizumab,efalizumab, muromab-CD3, natalizumab; TNF alpha Inhibitors such as forexample adalimumab, afelimomab, certolizumab pegol, golimumab,infliximab; Interleukin Inhibitors, basiliximab, canakinumab,daclizumab, mepolizumab, tocilizumab, ustekinumab; Radiopharmaceuticals,ibritumomab tiuxetan, tositumomab; Others Monoclonal Antibodies such asfor example abagovomab, adecatumumab, alemtuzumab, anti-CD30 monoclonalantibody Xmab2513, anti-MET monoclonal antibody MetMab, apolizumab,apomab, arcitumomab, basiliximab, bispecific antibody 2B1, blinatumomab,brentuximab vedotin, capromab pendetide, cixutumumab, claudiximab,conatumumab, dacetuzumab, denosumab, eculizumab, epratuzumab,epratuzumab, ertumaxomab, etaracizumab, figitumumab, fresolimumab,galiximab, ganitumab, gemtuzumab ozogamicin, glembatumumab, ibritumomab,inotuzumab ozogamicin, ipilimumab, lexatumumab, lintuzumab, lintuzumab,lucatumumab, mapatumumab, matuzumab, milatuzumab, monoclonal antibodyCC49, necitumumab, nimotuzumab, ofatumumab, oregovomab, pertuzumab,ramacurimab, ranibizumab, siplizumab, sonepcizumab, tanezumab,tositumomab, trastuzumab, tremelimumab, tucotuzumab celmoleukin,veltuzumab, visilizumab, volociximab, zalutumumab.

In some embodiments, the additional therapeutic agent is selected from:agents that affect the tumor micro-enviroment such as cellular signalingnetwork (e.g. phosphatidylinositol 3-kinase (PI3K) signaling pathway,signaling from the B-cell receptor and the IgE receptor). In someembodiments, the additional therapeutic agent is a PI3K signalinginhibitor or a syc kinase inhibitor. In one embodiment, the sykinhibitor is R788. In another embodiment is a PKCγ inhibitor such as byway of example only, enzastaurin.

Examples of agents that affect the tumor micro-environment include PI3Ksignaling inhibitor, syc kinase inhibitor, Protein Kinase Inhibitorssuch as for example dasatinib, erlotinib, everolimus, gefitinib,imatinib, lapatinib, nilotinib, pazonanib, sorafenib, sunitinib,temsirolimus; Other Angiogenesis Inhibitors such as for example GT-111,JI-101, R1530; Other Kinase Inhibitors such as for example AC220, AC480,ACE-041, AMG 900, AP24534, Any-614, AT7519, AT9283, AV-951, axitinib,AZD1152, AZD7762, AZD8055, AZD8931, bafetinib, BAY 73-4506, BGJ398,BGT226, BI 811283, BI6727, BIBF 1120, BIBW 2992, BMS-690154, BMS-777607,BMS-863233, BSK-461364, CAL-101, CEP-11981, CYC116, DCC-2036,dinaciclib, dovitinib lactate, E7050, EMD 1214063, ENMD-2076,fostamatinib disodium, GSK2256098, GSK690693, INCB18424, INNO-406,JNJ-26483327, JX-594, KX2-391, linifanib, LY2603618, MGCD265, MK-0457,MK1496, MLN8054, MLN8237, MP470, NMS-1116354, NMS-1286937, ON 01919.Na,OSI-027, OSI-930, Btk inhibitor, PF-00562271, PF-02341066, PF-03814735,PF-04217903, PF-04554878, PF-04691502, PF-3758309, PHA-739358, PLC3397,progenipoietin, R547, R763, ramucirumab, regorafenib, R05185426,SAR103168, SCH 727965, SGI-1176, SGX523, SNS-314, TAK-593, TAK-901,TKI258, TLN-232, TTP607, XL147, XL228, XL281RO5126766, XL418, XL765.

In some embodiments, the additional therapeutic agent is selected from:inhibitors of mitogen-activated protein kinase signaling, e.g., U0126,PD98059, PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY43-9006, wortmannin, or LY294002; Syk inhibitors; mTOR inhibitors; andantibodies (e.g., rituxan).

In some embodiments, the additional therapeutic agent is selected from:Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin, acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;altretamine; ambomycin; ametantrone acetate; aminoglutethimide;amsacrine; anastrozole; anthramycin; asparaginase; asperlin;azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide;bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycinsulfate; brequinar sodium; bropirimine; busulfan; cactinomycin;calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; daunorubicin hydrochloride; decitabine; dexormaplatin;dezaguanine; dezaguanine mesylate; diaziquone; doxorubicin; doxorubicinhydrochloride; droloxifene; droloxifene citrate; dromostanolonepropionate; duazomycin; edatrexate; eflornithine hydrochloride;elsamitrucin; enloplatin; enpromate; epipropidine; epirubicinhydrochloride; erbulozole; esorubicin hydrochloride; estramustine;estramustine phosphate sodium; etanidazole; etoposide; etoposidephosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide;floxuridine; fludarabine phosphate; fluorouracil; flurocitabine;fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride;hydroxyurea; idarubicin hydrochloride; ifosfamide; iimofosine;interleukin I1 (including recombinant interleukin II, or rlL2),interferon alfa-2a; interferon alfa-2b; interferon alfa-n1; interferonalfa-n3; interferon beta-1 a; interferon gamma-1 b; iproplatin;irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolideacetate; liarozole hydrochloride; lometrexol sodium; lomustine;losoxantrone hydrochloride; masoprocol; maytansine; mechlorethaminehydrochloride; megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; my cophenolicacid; nocodazoie; nogalamycin; ormaplatin; oxisuran; pegaspargase;peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman;piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimersodium; porfiromycin; prednimustine; procarbazine hydrochloride;puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide;safingol; safingol hydrochloride; semustine; simtrazene; sparfosatesodium; sparsomycin; spirogermanium hydrochloride; spiromustine;spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin;tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin;teniposide; teroxirone; testolactone; thiamiprine; thioguanine;thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestoloneacetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate;triptorelin; tubulozole hydrochloride; uracil mustard; uredepa;vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate;vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride.

In some embodiments, the additional therapeutic agent is selected from:20-epi-1, 25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; 9-dioxamycin; diphenyl spiromustine; docosanol;dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA;ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene;emitefur; epirubicin; epristeride; estramustine analogue; estrogenagonists; estrogen antagonists; etanidazole; etoposide phosphate;exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-such asfor example growth factor-1 receptor inhibitor; interferon agonists;interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen-binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer.

In some embodiments, the additional therapeutic agent is selected from:alkylating agents, antimetabolites, natural products, or hormones, e.g.,nitrogen mustards (e.g., mechloroethamine, cyclophosphamide,chlorambucil, etc.), alkyl sulfonates (e.g., busulfan), nitrosoureas(e.g., carmustine, lomusitne, ete.), or triazenes (decarbazine, etc.).Examples of antimetabolites include but are not limited to folic acidanalog (e.g., methotrexate), or pyrimidine analogs (e.g., Cytarabine),purine analogs (e.g., mercaptopurine, thioguanine, pentostatin).

In some embodiments, the additional therapeutic agent is selected from:nitrogen mustards (e.g., mechloroethamine, cyclophosphamide,chlorambucil, meiphalan, etc.), ethylenimine and methylmelamines (e.g.,hexamethlymelamine, thiotepa), alkyl sulfonates (e.g., busulfan),nitrosoureas (e.g., carmustine, lomusitne, semustine, streptozocin,etc.), or triazenes (decarbazine, ete.). Examples of antimetabolitesinclude, but are not limited to folic acid analog (e.g., methotrexate),or pyrimidine analogs (e.g., fluorouracil, floxouridine, Cytarabine),purine analogs (e.g., mercaptopurine, thioguanine, pentostatin.

In some embodiments, the additional therapeutic agent is selected from:agents which act by arresting cells in the G2-M phases due to stabilizedmicrotubules, e.g., Erbulozole (also known as R-55104), Dolastatin 10(also known as DLS-10 and NSC-376128), Mivobulin isethionate (also knownas CI-980), Vincristine, NSC-639829, Discodermolide (also known asNVP-XX-A-296), ABT-751 (Abbott, also known as E-7010), Altorhyrtins(such as Altorhyrtin A and Altorhyrtin C), Spongistatins (such asSpongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4,Spongistatin 5, Spongistatin 6, Spongistatin 7, Spongistatin 8, andSpongistatin 9), Cemadotin hydrochloride (also known as LU-103793 andNSC-D-669356), Epothilones (such as Epothilone A, Epothilone B,Epothilone C (also known as desoxyepothilone A or dEpoA), Epothilone D(also referred to as KOS-862, dEpoB, and desoxyepothilone B), EpothiloneE, Epothilone F, Epothilone B N-oxide, Epothilone A N-oxide,16-aza-epothilone B, 21-aminoepothilone B (also known as BMS-310705),21-hydroxyepothilone D (also known as Desoxyepothilone F and dEpoF),26-fluoroepothilone), Auristatin PE (also known as NSC-654663),Soblidotin (also known as TZT-1027), LS-4559-P (Pharmacia, also known asLS-4577), LS-4578 (Pharmacia, also known as LS-477-P), LS-4477(Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis), Vincristinesulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, also known asWS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (Hungarian Academy ofSciences), BSF-223651 (BASF, also known as ILX-651 and LU-223651),SAH-49960 (Lilly/Novartis), SDZ-268970 (Lilly/Novartis), AM-97(Armad/Kyowa Hakko), AM-132 (Armad), AM-138 (Armad/Kyowa Hakko),IDN-5005 (Indena), Cryptophycin 52 (also known as LY-355703), AC-7739(Ajinomoto, also known as AVE-8063A and CS-39.HCl), AC-7700 (Ajinomoto,also known as AVE-8062, AVE-8062A, CS-39-L-Ser.HCl, and RPR-258062A),Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (also known asNSC-106969), T-138067 (Tularik, also known as T-67, TL-138067 andTI-138067), COBRA-1 (Parker Hughes Institute, also known as DDE-261 andWHI-261), H10 (Kansas State University), H16 (Kansas State University),Oncocidin A1 (also known as BTO-956 and DIME), DDE-313 (Parker HughesInstitute), Fijianolide B, Laulimalide, SPA-2 (Parker Hughes Institute),SPA-1 (Parker Hughes Institute, also known as SPIKET-P), 3-IAABU(Cytoskeleton/Mt. Sinai School of Medicine, also known as MF-569),Narcosine (also known as NSC-5366), Nascapine, D-24851 (Asta Medica),A-105972 (Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton/Mt. Sinai Schoolof Medicine, also known as MF-191), TMPN (Arizona State University),Vanadocene acetylacetonate, T-138026 (Tularik), Monsatrol, lnanocine(also known as NSC-698666), 3-lAABE (Cytoskeleton/Mt. Sinai School ofMedicine), A-204197 (Abbott), T-607 (Tuiarik, also known as T-900607),RPR-115781 (Aventis), Eleutherobins (such as Desmethyleleutherobin,Desaetyleleutherobin, lsoeleutherobin A, and Z-Eleutherobin),Caribaeoside, Caribaeolin, Halichondrin B, D-64131 (Asta Medica),D-68144 (Asta Medica), Diazonamide A, A-293620 (Abbott), NPI-2350(Nereus), Taccalonolide A, TUB-245 (Aventis), A-259754 (Abbott),Diozostatin, (−)-Phenylahistin (also known as NSCL-96F037), D-68838(Asta Medica), D-68836 (Asta Medica), Myoseverin B, D-43411 (Zentaris,also known as D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-286(also known as SPA-110, trifluoroacetate salt) (Wyeth), D-82317(Zentaris), D-82318 (Zentaris), SC-12983 (NCI), Resverastatin phosphatesodium, BPR-OY-007 (National Health Research Institutes), and SSR-250411(Sanofi).

Pharmaceutical Compositions and Formulations

Disclosed herein, in certain embodiments, are pharmaceuticalcompositions and formulations comprising: (a) BTK inhibitor; (b) ananticancer agent, wherein the anticancer agent inhibits MALT1, MCL-1 orIDH1; and (c) a pharmaceutically-acceptable excipient. In someembodiment, the BTK inhibitor is ibrutinib. In some embodiments, theanticancer agent inhibits MALT1. In some embodiments, the anticanceragent that inhibits MALT1 comprises MI-2, mepazine, thioridazine, andpromazine. In some embodiments, the anticancer agent inhibits MCL-1. Insome embodiments, the anticancer agent that inhibits MCL-1 comprisesBI97C10, BI112D1, gossypol, obatoclax, MG-132, MIM1, sabutoclax, andTW-37. In some embodiments, the anticancer agent inhibits IDH1. In someembodiments, the anticancer agent that inhibits IDH1 comprises AGI-5198,AG-120, IDH-C227, and ML309. In some embodiments, the combination of aBTK inhibitor and an anticancer agent exert a synergistic effect. Insome embodiments, the combination of a BTK inhibitor and an anticanceragent exert an additive effect. In some embodiments, the combination ofa BTK inhibitor and an anticancer exert an antagonistic effect. In someembodiments, the combination of a BTK inhibitor and an anticancer agentsensitize cells to the BTK inhibitor. In some embodiments, thecombination of a BTK inhibitor and an anticancer agent exert no effecton the cells. In some embodiments, the BTK inhibitor is ibrutinib. Insome embodiments, the combination of ibrutinib and an anticancer agentexert a synergistic effect. In some embodiments, the combination ofibrutinib and an anticancer agent exert an additive effect. In someembodiments, the combination of ibrutinib and an anticancer agent exertan antagonistic effect. In some embodiments, the combination ofibrutinib and an anticancer agent sensitize cells to ibrutinib. In someembodiments, the combination of ibrutinib and an anticancer agent exertno effect on the cells. In some embodiments, a combination index (CI)value is used to indicate the behavior of the combination of ibrutiniband an anticancer agent.

In some embodiments, a pharmaceutical composition comprising: (1) a BTKinhibitor; a (b) a PIM inhibitor; and (c) a pharmaceutically-acceptableexcipient, is provided. An exemplary PIM inhibitor is AZD1208, and anexemplary BTK inhibitor is ibrutinib. In some embodiments, the PIMinhibitor is a PIM1 inhibitor. In some embodiments, the PIM inhibitor isa pan-PIM inhibitor. In some embodiments, the combination is in combineddosage form. In some embodiments, the combination is in separate dosageforms.

Pharmaceutical compositions may be formulated in a conventional mannerusing one or more physiologically acceptable carriers includingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen. Any ofthe well-known techniques, carriers, and excipients may be used assuitable and as understood in the art. A summary of pharmaceuticalcompositions described herein may be found, for example, in Remington:The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: MackPublishing Company, 1995); Hoover, John E., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems,Seventh Ed. (Lippincott Williams & Wilkins1999), herein incorporated byreference in their entirety.

A pharmaceutical composition, as used herein, refers to a mixture of acompound described herein, such as, for example, ibrutinib and ananticancer agent, with other chemical components, such as carriers,stabilizers, diluents, dispersing agents, suspending agents, thickeningagents, and/or excipients. The pharmaceutical composition facilitatesadministration of the compound to an organism. In practicing the methodsof treatment or use provided herein, therapeutically effective amountsof compounds described herein are administered in a pharmaceuticalcomposition to a mammal having a disease, disorder, or condition to betreated. Preferably, the mammal is a human. A therapeutically effectiveamount can vary widely depending on the severity of the disease, the ageand relative health of the subject, the potency of the compound used andother factors. The compounds can be used singly or in combination withone or more therapeutic agents as components of mixtures.

In certain embodiments, compositions may also include one or more pHadjusting agents or buffering agents, including acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids; bases such assodium hydroxide, sodium phosphate, sodium borate, sodium citrate,sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; andbuffers such as citrate/dextrose, sodium bicarbonate and ammoniumchloride. Such acids, bases and buffers are included in an amountrequired to maintain pH of the composition in an acceptable range.

In other embodiments, compositions may also include one or more salts inan amount required to bring osmolality of the composition into anacceptable range. Such salts include those having sodium, potassium orammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

The term “pharmaceutical combination” as used herein, means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. a compound described herein and a co-agent, are bothadministered to a patient simultaneously in the form of a single entityor dosage. The term “non-fixed combination” means that the activeingredients, e.g. a compound described herein and a co-agent, areadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific intervening time limits,wherein such administration provides effective levels of the twocompounds in the body of the patient. The latter also applies tococktail therapy, e.g. the administration of three or more activeingredients.

The pharmaceutical formulations described herein can be administered toa subject by multiple administration routes, including but not limitedto, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular),intranasal, buccal, topical, rectal, or transdermal administrationroutes. The pharmaceutical formulations described herein include, butare not limited to, aqueous liquid dispersions, self-emulsifyingdispersions, solid solutions, liposomal dispersions, aerosols, soliddosage forms, powders, immediate release formulations, controlledrelease formulations, fast melt formulations, tablets, capsules, pills,delayed release formulations, extended release formulations, pulsatilerelease formulations, multiparticulate formulations, and mixed immediateand controlled release formulations.

Pharmaceutical compositions including a compound described herein may bemanufactured in a conventional manner, such as, by way of example only,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or compressionprocesses.

“Antifoaming agents” reduce foaming during processing which can resultin coagulation of aqueous dispersions, bubbles in the finished film, orgenerally impair processing. Exemplary anti-foaming agents includesilicon emulsions or sorbitan sesquoleate.

“Antioxidants” include, for example, butylated hydroxytoluene (BHT),sodium ascorbate, ascorbic acid, sodium metabisulfite and tocopherol. Incertain embodiments, antioxidants enhance chemical stability whererequired.

In certain embodiments, compositions provided herein may also includeone or more preservatives to inhibit microbial activity. Suitablepreservatives include mercury-containing substances such as merfen andthiomersal; stabilized chlorine dioxide; and quaternary ammoniumcompounds such as benzalkonium chloride, cetyltrimethylammonium bromideand cetylpyridinium chloride.

Formulations described herein may benefit from antioxidants, metalchelating agents, thiol containing compounds and other generalstabilizing agents. Examples of such stabilizing agents, include, butare not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/vmonothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% toabout 2% w/v ascorbic acid, (0.003% to about 0.02% w/v polysorbate 80,(g) 0.001% to about 0.05% w/v. polysorbate 20, (h) arginine, (i)heparin, (j) dextran sulfate, (k) cyclodextrins, (1) pentosanpolysulfate and other heparinoids, (m) divalent cations such asmagnesium and zinc; or (n) combinations thereof.

“Binders” impart cohesive qualities and include, e.g., alginic acid andsalts thereof; cellulose derivatives such as carboxymethylcellulose,methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose,hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel®),ethylcellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g.,Avicel®); microcrystalline dextrose; amylose; magnesium aluminumsilicate; polysaccharide acids; bentonites; gelatin;polyvinylpyrrolidone/vinyl acetate copolymer; crospovidone; povidone;starch; pregelatinized starch; tragacanth, dextrin, a sugar, such assucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol,xylitol (e.g., Xylitab®), and lactose; a natural or synthetic gum suchas acacia, tragacanth, ghatti gum, mucilage of isapol husks,polyvinylpyrrolidone (e.g., Polyvidone® CL, Kollidon® CL, Polyplasdone®XL-10), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodiumalginate, and the like.

A “carrier” or “carrier materials” include any commonly used excipientsin pharmaceutics and should be selected on the basis of compatibilitywith compounds disclosed herein, such as, compounds of ibrutinib and Ananticancer agent, and the release profile properties of the desireddosage form. Exemplary carrier materials include, e.g., binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, diluents, and thelike. “Pharmaceutically compatible carrier materials” may include, butare not limited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like. See,e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed(Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins1999).

“Dispersing agents,” and/or “viscosity modulating agents” includematerials that control the diffusion and homogeneity of a drug throughliquid media or a granulation method or blend method. In someembodiments, these agents also facilitate the effectiveness of a coatingor eroding matrix. Exemplary diffusion facilitators/dispersing agentsinclude, e.g., hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG,polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and thecarbohydrate-based dispersing agents such as, for example, hydroxypropylcelluloses (e.g., HPC, HPC-SL, and HPC-L), hydroxypropylmethylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M),carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), noncrystallinecellulose, magnesium aluminum silicate, triethanolamine, polyvinylalcohol (PVA), vinyl pyrrolidone/vinyl acetate copolymer (S630),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol), poloxamers (e.g., PluronicsF68®, F88®, and F108®, which are block copolymers of ethylene oxide andpropylene oxide); and poloxamines (e.g., Tetronic 908®, also known asPoloxamine 908®, which is a tetrafunctional block copolymer derived fromsequential addition of propylene oxide and ethylene oxide toethylenediamine (BASF Corporation, Parsippany, N.J.)),polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetatecopolymer (S-630), polyethylene glycol, e.g., the polyethylene glycolcan have a molecular weight of about 300 to about 6000, or about 3350 toabout 4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g.,gum tragacanth and gum acacia, guar gum, xanthans, including xanthangum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose,methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodiumalginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitanmonolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates,chitosans and combinations thereof. Plasticizers such as cellulose ortriethyl cellulose can also be used as dispersing agents. Dispersingagents particularly useful in liposomal dispersions and self-emulsifyingdispersions are dimyristoyl phosphatidyl choline, natural phosphatidylcholine from eggs, natural phosphatidyl glycerol from eggs, cholesteroland isopropyl myristate.

Combinations of one or more erosion facilitator with one or morediffusion facilitator can also be used in the present compositions.

The term “diluent” refers to chemical compounds that are used to dilutethe compound of interest prior to delivery. Diluents can also be used tostabilize compounds because they can provide a more stable environment.Salts dissolved in buffered solutions (which also can provide pH controlor maintenance) are utilized as diluents in the art, including, but notlimited to a phosphate buffered saline solution. In certain embodiments,diluents increase bulk of the composition to facilitate compression orcreate sufficient bulk for homogenous blend for capsule filling. Suchcompounds include e.g., lactose, starch, mannitol, sorbitol, dextrose,microcrystalline cellulose such as Avicel®; dibasic calcium phosphate,dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate;anhydrous lactose, spray-dried lactose; pregelatinized starch,compressible sugar, such as Di-Pac® (Amstar); mannitol,hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetatestearate, sucrose-based diluents, confectioner's sugar; monobasiccalcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactatetrihydrate, dextrates; hydrolyzed cereal solids, amylose; powderedcellulose, calcium carbonate; glycine, kaolin; mannitol, sodiumchloride; inositol, bentonite, and the like.

The term “disintegrate” includes both the dissolution and dispersion ofthe dosage form when contacted with gastrointestinal fluid.“Disintegration agents or disintegrants” facilitate the breakup ordisintegration of a substance. Examples of disintegration agents includea starch, e.g., a natural starch such as corn starch or potato starch, apregelatinized starch such as National 1551 or Amijel®, or sodium starchglycolate such as Promogel® or Explotab®, a cellulose such as a woodproduct, methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101,Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, MingTia®, and Solka-Floc®, methylcellulose, croscarmellose, or across-linked cellulose, such as cross-linked sodiumcarboxymethylcellulose (Ac-Di-Sol®), cross-linkedcarboxymethylcellulose, or cross-linked croscarmellose, a cross-linkedstarch such as sodium starch glycolate, a cross-linked polymer such ascrospovidone, a cross-linked polyvinylpyrrolidone, alginate such asalginic acid or a salt of alginic acid such as sodium alginate, a claysuch as Veegum® HV (magnesium aluminum silicate), a gum such as agar,guar, locust bean, Karaya, pectin, or tragacanth, sodium starchglycolate, bentonite, a natural sponge, a surfactant, a resin such as acation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium laurylsulfate in combination starch, and the like.

“Drug absorption” or “absorption” typically refers to the process ofmovement of drug from site of administration of a drug across a barrierinto a blood vessel or the site of action, e.g., a drug moving from thegastrointestinal tract into the portal vein or lymphatic system.

An “enteric coating” is a substance that remains substantially intact inthe stomach but dissolves and releases the drug in the small intestineor colon. Generally, the enteric coating comprises a polymeric materialthat prevents release in the low pH environment of the stomach but thationizes at a higher pH, typically a pH of 6 to 7, and thus dissolvessufficiently in the small intestine or colon to release the active agenttherein.

“Erosion facilitators” include materials that control the erosion of aparticular material in gastrointestinal fluid. Erosion facilitators aregenerally known to those of ordinary skill in the art. Exemplary erosionfacilitators include, e.g., hydrophilic polymers, electrolytes,proteins, peptides, and amino acids.

“Filling agents” include compounds such as lactose, calcium carbonate,calcium phosphate, dibasic calcium phosphate, calcium sulfate,microcrystalline cellulose, cellulose powder, dextrose, dextrates,dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol,mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

“Flavoring agents” and/or “sweeteners” useful in the formulationsdescribed herein, include, e.g., acacia syrup, acesulfame K, alitame,anise, apple, aspartame, banana, Bavarian cream, berry, black currant,butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream,chocolate, cinnamon, bubble gum, citrus, citrus punch, citrus cream,cotton candy, cocoa, cola, cool cherry, cool citrus, cyclamate,cylamate, dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger,glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit, honey,isomalt, lemon, lime, lemon cream, monoammonium glyrrhizinate(MagnaSweet®), maltol, mannitol, maple, marshmallow, menthol, mintcream, mixed berry, neohesperidine DC, neotame, orange, pear, peach,peppermint, peppermint cream, Prosweet® Powder, raspberry, root beer,rum, saccharin, safrole, sorbitol, spearmint, spearmint cream,strawberry, strawberry cream, stevia, sucralose, sucrose, sodiumsaccharin, saccharin, aspartame, acesulfame potassium, mannitol, talin,sylitol, sucralose, sorbitol, Swiss cream, tagatose, tangerine,thaumatin, tutti fruitti, vanilla, walnut, watermelon, wild cherry,wintergreen, xylitol, or any combination of these flavoring ingredients,e.g., anise-menthol, cherry-anise, cinnamon-orange, cherry-cinnamon,chocolate-mint, honey-lemon, lemon-lime, lemon-mint, menthol-eucalyptus,orange-cream, vanilla-mint, and mixtures thereof.

“Lubricants” and “glidants” are compounds that prevent, reduce orinhibit adhesion or friction of materials. Exemplary lubricants include,e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, ahydrocarbon such as mineral oil, or hydrogenated vegetable oil such ashydrogenated soybean oil (Sterotex®), higher fatty acids and theiralkali-metal and alkaline earth metal salts, such as aluminum, calcium,magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes,Stearowet®, boric acid, sodium benzoate, sodium acetate, sodiumchloride, leucine, a polyethylene glycol (e.g., PEG-4000) or amethoxypolyethylene glycol such as Carbowax™, sodium oleate, sodiumbenzoate, glyceryl behenate, polyethylene glycol, magnesium or sodiumlauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil®, a starchsuch as corn starch, silicone oil, a surfactant, and the like.

A “measurable serum concentration” or “measurable plasma concentration”describes the blood serum or blood plasma concentration, typicallymeasured in mg, μg, or ng of therapeutic agent per mL, dL, or L of bloodserum, absorbed into the bloodstream after administration. As usedherein, measurable plasma concentrations are typically measured in ng/mlor μg/ml.

“Pharmacodynamics” refers to the factors which determine the biologicresponse observed relative to the concentration of drug at a site ofaction.

“Pharmacokinetics” refers to the factors which determine the attainmentand maintenance of the appropriate concentration of drug at a site ofaction.

“Plasticizers” are compounds used to soften the microencapsulationmaterial or film coatings to make them less brittle. Suitableplasticizers include, e.g., polyethylene glycols such as PEG 300, PEG400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propyleneglycol, oleic acid, triethyl cellulose and triacetin. In someembodiments, plasticizers can also function as dispersing agents orwetting agents.

“Solubilizers” include compounds such as triacetin, triethylcitrate,ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate,vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone,N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropylalcohol, cholesterol, bile salts, polyethylene glycol 200-600,glycofurol, transcutol, propylene glycol, and dimethyl isosorbide andthe like.

“Stabilizers” include compounds such as any antioxidation agents,buffers, acids, preservatives and the like.

“Steady state,” as used herein, is when the amount of drug administeredis equal to the amount of drug eliminated within one dosing intervalresulting in a plateau or constant plasma drug exposure.

“Suspending agents” include compounds such as polyvinylpyrrolidone,e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17,polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g.,the polyethylene glycol can have a molecular weight of about 300 toabout 6000, or about 3350 to about 4000, or about 7000 to about 5400,sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate,polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as,e.g., gum tragacanth and gum acacia, guar gum, xanthans, includingxanthan gum, sugars, cellulosics, such as, e.g., sodiumcarboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80,sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylatedsorbitan monolaurate, povidone and the like.

“Surfactants” include compounds such as sodium lauryl sulfate, sodiumdocusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitanmonooleate, polyoxyethylene sorbitan monooleate, polysorbates,polaxomers, bile salts, glyceryl monostearate, copolymers of ethyleneoxide and propylene oxide, e.g., Pluronic® (BASF), and the like. Someother surfactants include polyoxyethylene fatty acid glycerides andvegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40. In some embodiments, surfactants may be included toenhance physical stability or for other purposes.

“Viscosity enhancing agents” include, e.g., methyl cellulose, xanthangum, carboxymethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetatestearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinylalcohol, alginates, acacia, chitosans and combinations thereof.

“Wetting agents” include compounds such as oleic acid, glycerylmonostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamineoleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate,sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium saltsand the like.

Dosage Forms

The compositions described herein can be formulated for administrationto a subject via any conventional means including, but not limited to,oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular),buccal, intranasal, rectal or transdermal administration routes. In someembodiments, the composition is formulated for administration in acombined dosage form. In some embodiments, the composition is formulatedfor administration in a separate dosage forms. As used herein, the term“subject” is used to mean an animal, preferably a mammal, including ahuman or non-human. The terms “individual(s)”, “subject(s)” and“patient(s)” are used interchangeably herein, and mean any mammal. Insome embodiments, the mammal is a human. In some embodiments, the mammalis a non-human. None of the terms require or are limited to situationscharacterized by the supervision (e.g. constant or intermittent) of ahealth care worker (e.g. a doctor, a registered nurse, a nursepractitioner, a physician's assistant, an orderly or a hospice worker).

Moreover, the pharmaceutical compositions described herein, whichinclude ibrutinib and/or an anticancer agent can be formulated into anysuitable dosage form, including but not limited to, aqueous oraldispersions, liquids, gels, syrups, elixirs, slurries, suspensions andthe like, for oral ingestion by a patient to be treated, solid oraldosage forms, aerosols, controlled release formulations, fast meltformulations, effervescent formulations, lyophilized formulations,tablets, powders, pills, dragees, capsules, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, multiparticulate formulations, and mixed immediate releaseand controlled release formulations.

Pharmaceutical preparations for oral use can be obtained by mixing oneor more solid excipient with one or more of the compounds describedherein, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients include, forexample, fillers such as sugars, including lactose, sucrose, mannitol,or sorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methylcellulose, microcrystalline cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or otherssuch as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. Ifdesired, disintegrating agents may be added, such as the cross-linkedcroscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or asalt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

In some embodiments, the solid dosage forms disclosed herein may be inthe form of a tablet, (including a suspension tablet, a fast-melttablet, a bite-disintegration tablet, a rapid-disintegration tablet, aneffervescent tablet, or a caplet), a pill, a powder (including a sterilepackaged powder, a dispensable powder, or an effervescent powder) acapsule (including both soft or hard capsules, e.g., capsules made fromanimal-derived gelatin or plant-derived HPMC, or “sprinkle capsules”),solid dispersion, solid solution, bioerodible dosage form, controlledrelease formulations, pulsatile release dosage forms, multiparticulatedosage forms, pellets, granules, or an aerosol. In other embodiments,the pharmaceutical formulation is in the form of a powder. In stillother embodiments, the pharmaceutical formulation is in the form of atablet, including but not limited to, a fast-melt tablet. Additionally,pharmaceutical formulations described herein may be administered as asingle capsule or in multiple capsule dosage form. In some embodiments,the pharmaceutical formulation is administered in two, or three, orfour, capsules or tablets.

In some embodiments, solid dosage forms, e.g., tablets, effervescenttablets, and capsules, are prepared by mixing particles of ibrutiniband/or an anticancer agent, with one or more pharmaceutical excipientsto form a bulk blend composition. When referring to these bulk blendcompositions as homogeneous, it is meant that the particles of ibrutiniband/or an anticancer agent, are dispersed evenly throughout thecomposition so that the composition may be readily subdivided intoequally effective unit dosage forms, such as tablets, pills, andcapsules. The individual unit dosages may also include film coatings,which disintegrate upon oral ingestion or upon contact with diluent.These formulations can be manufactured by conventional pharmacologicaltechniques.

Conventional pharmacological techniques include, e.g., one or acombination of methods: (1) dry mixing, (2) direct compression, (3)milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6)fusion. See, e.g., Lachman et al., The Theory and Practice of IndustrialPharmacy (1986). Other methods include, e.g., spray drying, pan coating,melt granulation, granulation, fluidized bed spray drying or coating(e.g., wurster coating), tangential coating, top spraying, tableting,extruding and the like.

The pharmaceutical solid dosage forms described herein can include acompound described herein and one or more pharmaceutically acceptableadditives such as a compatible carrier, binder, filling agent,suspending agent, flavoring agent, sweetening agent, disintegratingagent, dispersing agent, surfactant, lubricant, colorant, diluent,solubilizer, moistening agent, plasticizer, stabilizer, penetrationenhancer, wetting agent, anti-foaming agent, antioxidant, preservative,or one or more combination thereof. In still other aspects, usingstandard coating procedures, such as those described in Remington'sPharmaceutical Sciences, 20th Edition (2000), a film coating is providedaround the formulation of ibrutinib and/or an anticancer agent. Inanother embodiment, some or all of the particles of ibrutinib and/or ananticancer agent, are not microencapsulated and are uncoated.

Suitable carriers for use in the solid dosage forms described hereininclude, but are not limited to, acacia, gelatin, colloidal silicondioxide, calcium glycerophosphate, calcium lactate, maltodextrin,glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodiumchloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyllactylate, carrageenan, monoglyceride, diglyceride, pregelatinizedstarch, hydroxypropylmethylcellulose, hydroxypropylmethylcelluloseacetate stearate, sucrose, microcrystalline cellulose, lactose, mannitoland the like.

Suitable filling agents for use in the solid dosage forms describedherein include, but are not limited to, lactose, calcium carbonate,calcium phosphate, dibasic calcium phosphate, calcium sulfate,microcrystalline cellulose, cellulose powder, dextrose, dextrates,dextran, starches, pregelatinized starch, hydroxypropylmethycellulose(HPMC), hydroxypropylmethycellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), sucrose,xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethyleneglycol, and the like.

In order to release the compound of ibrutinib and/or an anticanceragent, from a solid dosage form matrix as efficiently as possible,disintegrants are often used in the formulation, especially when thedosage forms are compressed with binder. Disintegrants help rupturingthe dosage form matrix by swelling or capillary action when moisture isabsorbed into the dosage form. Suitable disintegrants for use in thesolid dosage forms described herein include, but are not limited to,natural starch such as corn starch or potato starch, a pregelatinizedstarch such as National 1551 or Amijel®, or sodium starch glycolate suchas Promogel® or Explotab®, a cellulose such as a wood product,methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel®PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, andSolka-Floc®, methylcellulose, croscarmellose, or a cross-linkedcellulose, such as cross-linked sodium carboxymethylcellulose(Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linkedcroscarmellose, a cross-linked starch such as sodium starch glycolate, across-linked polymer such as crospovidone, a cross-linkedpolyvinylpyrrolidone, alginate such as alginic acid or a salt of alginicacid such as sodium alginate, a clay such as Veegum® HV (magnesiumaluminum silicate), a gum such as agar, guar, locust bean, Karaya,pectin, or tragacanth, sodium starch glycolate, bentonite, a naturalsponge, a surfactant, a resin such as a cation-exchange resin, citruspulp, sodium lauryl sulfate, sodium lauryl sulfate in combinationstarch, and the like.

Binders impart cohesiveness to solid oral dosage form formulations: forpowder filled capsule formulation, they aid in plug formation that canbe filled into soft or hard shell capsules and for tablet formulation,they ensure the tablet remaining intact after compression and helpassure blend uniformity prior to a compression or fill step. Materialssuitable for use as binders in the solid dosage forms described hereininclude, but are not limited to, carboxymethylcellulose, methylcellulose(e.g., Methocel®), hydroxypropylmethylcellulose (e.g. Hypromellose USPPharmacoat-603, hydroxypropylmethylcellulose acetate stearate (AqoateHS-LF and HS), hydroxyethylcellulose, hydroxypropylcellulose (e.g.,Klucel®), ethylcellulose (e.g., Ethocel®), and microcrystallinecellulose (e.g., Avicel®), microcrystalline dextrose, amylose, magnesiumaluminum silicate, polysaccharide acids, bentonites, gelatin,polyvinylpyrrolidone/vinyl acetate copolymer, crospovidone, povidone,starch, pregelatinized starch, tragacanth, dextrin, a sugar, such assucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol,xylitol (e.g., Xylitab®), lactose, a natural or synthetic gum such asacacia, tragacanth, ghatti gum, mucilage of isapol husks, starch,polyvinylpyrrolidone (e.g., Povidone® CL, Kollidon® CL, Polyplasdone®XL-10, and Povidone® K-12), larch arabogalactan, Veegum®, polyethyleneglycol, waxes, sodium alginate, and the like.

In general, binder levels of 20-70% are used in powder-filled gelatincapsule formulations. Binder usage level in tablet formulations varieswhether direct compression, wet granulation, roller compaction, or usageof other excipients such as fillers which itself can act as moderatebinder. Formulators skilled in art can determine the binder level forthe formulations, but binder usage level of up to 70% in tabletformulations is common.

Suitable lubricants or glidants for use in the solid dosage formsdescribed herein include, but are not limited to, stearic acid, calciumhydroxide, talc, corn starch, sodium stearyl fumerate, alkali-metal andalkaline earth metal salts, such as aluminum, calcium, magnesium, zinc,stearic acid, sodium stearates, magnesium stearate, zinc stearate,waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodiumchloride, leucine, a polyethylene glycol or a methoxypolyethylene glycolsuch as Carbowax™, PEG 4000, PEG 5000, PEG 6000, propylene glycol,sodium oleate, glyceryl behenate, glyceryl palmitostearate, glycerylbenzoate, magnesium or sodium lauryl sulfate, and the like.

Suitable diluents for use in the solid dosage forms described hereininclude, but are not limited to, sugars (including lactose, sucrose, anddextrose), polysaccharides (including dextrates and maltodextrin),polyols (including mannitol, xylitol, and sorbitol), cyclodextrins andthe like.

The term “non water-soluble diluent” represents compounds typically usedin the formulation of pharmaceuticals, such as calcium phosphate,calcium sulfate, starches, modified starches and microcrystallinecellulose, and microcellulose (e.g., having a density of about 0.45g/cm³, e.g. Avicel, powdered cellulose), and talc.

Suitable wetting agents for use in the solid dosage forms describedherein include, for example, oleic acid, glyceryl monostearate, sorbitanmonooleate, sorbitan monolaurate, triethanolamine oleate,polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, quaternary ammonium compounds (e.g., Polyquat 10®), sodiumoleate, sodium lauryl sulfate, magnesium stearate, sodium docusate,triacetin, vitamin E TPGS and the like.

Suitable surfactants for use in the solid dosage forms described hereininclude, for example, sodium lauryl sulfate, sorbitan monooleate,polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bilesalts, glyceryl monostearate, copolymers of ethylene oxide and propyleneoxide, e.g., Pluronic® (BASF), and the like.

Suitable suspending agents for use in the solid dosage forms describedhere include, but are not limited to, polyvinylpyrrolidone, e.g.,polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyethylene glycol, e.g., thepolyethylene glycol can have a molecular weight of about 300 to about6000, or about 3350 to about 4000, or about 7000 to about 5400, vinylpyrrolidone/vinyl acetate copolymer (S630), sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as,e.g., gum tragacanth and gum acacia, guar gum, xanthans, includingxanthan gum, sugars, cellulosics, such as, e.g., sodiumcarboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80,sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylatedsorbitan monolaurate, povidone and the like.

Suitable antioxidants for use in the solid dosage forms described hereininclude, for example, e.g., butylated hydroxytoluene (BHT), sodiumascorbate, and tocopherol.

It should be appreciated that there is considerable overlap betweenadditives used in the solid dosage forms described herein. Thus, theabove-listed additives should be taken as merely exemplary, and notlimiting, of the types of additives that can be included in solid dosageforms described herein. The amounts of such additives can be readilydetermined by one skilled in the art, according to the particularproperties desired.

In other embodiments, one or more layers of the pharmaceuticalformulation are plasticized. Illustratively, a plasticizer is generallya high boiling point solid or liquid. Suitable plasticizers can be addedfrom about 0.01% to about 50% by weight (w/w) of the coatingcomposition. Plasticizers include, but are not limited to, diethylphthalate, citrate esters, polyethylene glycol, glycerol, acetylatedglycerides, triacetin, polypropylene glycol, polyethylene glycol,triethyl citrate, dibutyl sebacate, stearic acid, stearol, stearate, andcastor oil.

Compressed tablets are solid dosage forms prepared by compacting thebulk blend of the formulations described above. In various embodiments,compressed tablets which are designed to dissolve in the mouth willinclude one or more flavoring agents. In other embodiments, thecompressed tablets will include a film surrounding the final compressedtablet. In some embodiments, the film coating can provide a delayedrelease of ibrutinib or the second agent, from the formulation. In otherembodiments, the film coating aids in patient compliance (e.g., Opadry®coatings or sugar coating). Film coatings including Opadry® typicallyrange from about 1% to about 3% of the tablet weight. In otherembodiments, the compressed tablets include one or more excipients.

A capsule may be prepared, for example, by placing the bulk blend of theformulation of ibrutinib or the second agent, described above, inside ofa capsule. In some embodiments, the formulations (non-aqueoussuspensions and solutions) are placed in a soft gelatin capsule. Inother embodiments, the formulations are placed in standard gelatincapsules or non-gelatin capsules such as capsules comprising HPMC. Inother embodiments, the formulation is placed in a sprinkle capsule,wherein the capsule may be swallowed whole or the capsule may be openedand the contents sprinkled on food prior to eating. In some embodiments,the therapeutic dose is split into multiple (e.g., two, three, or four)capsules. In some embodiments, the entire dose of the formulation isdelivered in a capsule form.

In various embodiments, the particles of ibrutinib and/or an anticanceragent, and one or more excipients are dry blended and compressed into amass, such as a tablet, having a hardness sufficient to provide apharmaceutical composition that substantially disintegrates within lessthan about 30 minutes, less than about 35 minutes, less than about 40minutes, less than about 45 minutes, less than about 50 minutes, lessthan about 55 minutes, or less than about 60 minutes, after oraladministration, thereby releasing the formulation into thegastrointestinal fluid.

In another aspect, dosage forms may include microencapsulatedformulations. In some embodiments, one or more other compatiblematerials are present in the microencapsulation material. Exemplarymaterials include, but are not limited to, pH modifiers, erosionfacilitators, anti-foaming agents, antioxidants, flavoring agents, andcarrier materials such as binders, suspending agents, disintegrationagents, filling agents, surfactants, solubilizers, stabilizers,lubricants, wetting agents, and diluents.

Materials useful for the microencapsulation described herein includematerials compatible with ibrutinib and/or an anticancer agent, whichsufficiently isolate the compound of any of ibrutinib or an anticanceragent, from other non-compatible excipients. Materials compatible withcompounds of any of ibrutinib or an anticancer agent, are those thatdelay the release of the compounds of any of ibrutinib or an anticanceragent, in vivo.

Exemplary microencapsulation materials useful for delaying the releaseof the formulations including compounds described herein, include, butare not limited to, hydroxypropyl cellulose ethers (HPC) such as Klucel®or Nisso HPC, low-substituted hydroxypropyl cellulose ethers (L-HPC),hydroxypropyl methyl cellulose ethers (HPMC) such as Seppifilm-LC,Pharmacoat®, Metolose SR, Methocel®-E, Opadry YS, PrimaFlo, BenecelMP824, and Benecel MP843, methylcellulose polymers such as Methocel®-A,hydroxypropylmethylcellulose acetate stearate Aqoat (HF-LS, HF-LG,HF-MS)and Metolose®, Ethylcelluloses (EC) and mixtures thereof such as E461,Ethocel®, Aqualon®-EC, Surelease®, Polyvinyl alcohol (PVA) such asOpadry AMB, hydroxyethylcelluloses such as Natrosol®,carboxymethylcelluloses and salts of carboxymethylcelluloses (CMC) suchas Aqualon®-CMC, polyvinyl alcohol and polyethylene glycol co-polymerssuch as Kollicoat IR®, monoglycerides (Myverol), triglycerides (KLX),polyethylene glycols, modified food starch, acrylic polymers andmixtures of acrylic polymers with cellulose ethers such as Eudragit®EPO, Eudragit® L30D-55, Eudragit® FS 30D Eudragit® L100-55, Eudragit®L100, Eudragit® 5100, Eudragit® RD100, Eudragit® E100, Eudragit® L12.5,Eudragit® 512.5, Eudragit® NE30D, and Eudragit® NE 40D, celluloseacetate phthalate, sepifilms such as mixtures of HPMC and stearic acid,cyclodextrins, and mixtures of these materials.

In still other embodiments, plasticizers such as polyethylene glycols,e.g., PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800,stearic acid, propylene glycol, oleic acid, and triacetin areincorporated into the microencapsulation material. In other embodiments,the microencapsulating material useful for delaying the release of thepharmaceutical compositions is from the USP or the National Formulary(NF). In yet other embodiments, the microencapsulation material isKlucel. In still other embodiments, the microencapsulation material ismethocel.

Microencapsulated compounds of any of ibrutinib or an anticancer agentmay be formulated by methods known by one of ordinary skill in the art.Such known methods include, e.g., spray drying processes, spinningdisk-solvent processes, hot melt processes, spray chilling methods,fluidized bed, electrostatic deposition, centrifugal extrusion,rotational suspension separation, polymerization at liquid-gas orsolid-gas interface, pressure extrusion, or spraying solvent extractionbath. In addition to these, several chemical techniques, e.g., complexcoacervation, solvent evaporation, polymer-polymer incompatibility,interfacial polymerization in liquid media, in situ polymerization,in-liquid drying, and desolvation in liquid media could also be used.Furthermore, other methods such as roller compaction,extrusion/spheronization, coacervation, or nanoparticle coating may alsobe used.

In one embodiment, the particles of compounds of any of ibrutinib or ananticancer agent are microencapsulated prior to being formulated intoone of the above forms. In still another embodiment, some or most of theparticles are coated prior to being further formulated by using standardcoating procedures, such as those described in Remington'sPharmaceutical Sciences, 20th Edition (2000).

In other embodiments, the solid dosage formulations of the compounds ofany of ibrutinib and/or an anticancer agent are plasticized (coated)with one or more layers. Illustratively, a plasticizer is generally ahigh boiling point solid or liquid. Suitable plasticizers can be addedfrom about 0.01% to about 50% by weight (w/w) of the coatingcomposition. Plasticizers include, but are not limited to, diethylphthalate, citrate esters, polyethylene glycol, glycerol, acetylatedglycerides, triacetin, polypropylene glycol, polyethylene glycol,triethyl citrate, dibutyl sebacate, stearic acid, stearol, stearate, andcastor oil.

In other embodiments, a powder including the formulations with acompound of any of ibrutinib and/or an anticancer agent, describedherein, may be formulated to include one or more pharmaceuticalexcipients and flavors. Such a powder may be prepared, for example, bymixing the formulation and optional pharmaceutical excipients to form abulk blend composition. Additional embodiments also include a suspendingagent and/or a wetting agent. This bulk blend is uniformly subdividedinto unit dosage packaging or multi-dosage packaging units.

In still other embodiments, effervescent powders are also prepared inaccordance with the present disclosure. Effervescent salts have beenused to disperse medicines in water for oral administration.Effervescent salts are granules or coarse powders containing a medicinalagent in a dry mixture, usually composed of sodium bicarbonate, citricacid and/or tartaric acid. When salts of the compositions describedherein are added to water, the acids and the base react to liberatecarbon dioxide gas, thereby causing “effervescence.” Examples ofeffervescent salts include, e.g., the following ingredients: sodiumbicarbonate or a mixture of sodium bicarbonate and sodium carbonate,citric acid and/or tartaric acid. Any acid-base combination that resultsin the liberation of carbon dioxide can be used in place of thecombination of sodium bicarbonate and citric and tartaric acids, as longas the ingredients were suitable for pharmaceutical use and result in apH of about 6.0 or higher.

In some embodiments, the solid dosage forms described herein can beformulated as enteric coated delayed release oral dosage forms, i.e., asan oral dosage form of a pharmaceutical composition as described hereinwhich utilizes an enteric coating to affect release in the smallintestine of the gastrointestinal tract. The enteric coated dosage formmay be a compressed or molded or extruded tablet/mold (coated oruncoated) containing granules, powder, pellets, beads or particles ofthe active ingredient and/or other composition components, which arethemselves coated or uncoated. The enteric coated oral dosage form mayalso be a capsule (coated or uncoated) containing pellets, beads orgranules of the solid carrier or the composition, which are themselvescoated or uncoated.

The term “delayed release” as used herein refers to the delivery so thatthe release can be accomplished at some generally predictable locationin the intestinal tract more distal to that which would have beenaccomplished if there had been no delayed release alterations. In someembodiments the method for delay of release is coating. Any coatingsshould be applied to a sufficient thickness such that the entire coatingdoes not dissolve in the gastrointestinal fluids at pH below about 5,but does dissolve at pH about 5 and above. It is expected that anyanionic polymer exhibiting a pH-dependent solubility profile can be usedas an enteric coating in the methods and compositions described hereinto achieve delivery to the lower gastrointestinal tract. In someembodiments the polymers described herein are anionic carboxylicpolymers. In other embodiments, the polymers and compatible mixturesthereof, and some of their properties, include, but are not limited to:

Shellac, also called purified lac, a refined product obtained from theresinous secretion of an insect. This coating dissolves in media ofpH>7;

Acrylic polymers. The performance of acrylic polymers (primarily theirsolubility in biological fluids) can vary based on the degree and typeof substitution. Examples of suitable acrylic polymers includemethacrylic acid copolymers and ammonium methacrylate copolymers. TheEudragit series E, L, S, RL, RS and NE (Rohm Pharma) are available assolubilized in organic solvent, aqueous dispersion, or dry powders. TheEudragit series RL, NE, and RS are insoluble in the gastrointestinaltract but are permeable and are used primarily for colonic targeting.The Eudragit series E dissolve in the stomach. The Eudragit series L,L-30D and S are insoluble in stomach and dissolve in the intestine;

Cellulose Derivatives. Examples of suitable cellulose derivatives are:ethyl cellulose; reaction mixtures of partial acetate esters ofcellulose with phthalic anhydride. The performance can vary based on thedegree and type of substitution. Cellulose acetate phthalate (CAP)dissolves in pH>6. Aquateric (FMC) is an aqueous based system and is aspray dried CAP psuedolatex with particles <1 μm. Other components inAquateric can include pluronics, Tweens, and acetylated monoglycerides.Other suitable cellulose derivatives include: cellulose acetatetrimellitate (Eastman); methylcellulose (Pharmacoat, Methocel);hydroxypropylmethyl cellulose phthalate (HPMCP); hydroxypropylmethylcellulose succinate (HPMCS); and hydroxypropylmethylcellulose acetatesuccinate (e.g., AQOAT (Shin Etsu)). The performance can vary based onthe degree and type of substitution. For example, HPMCP such as, HP-50,HP-55, HP-55S, HP-55F grades are suitable. The performance can varybased on the degree and type of substitution. For example, suitablegrades of hydroxypropylmethylcellulose acetate succinate include, butare not limited to, AS-LG (LF), which dissolves at pH 5, AS-MG (MF),which dissolves at pH 5.5, and AS-HG (HF), which dissolves at higher pH.These polymers are offered as granules, or as fine powders for aqueousdispersions; Poly Vinyl Acetate Phthalate (PVAP). PVAP dissolves inpH>5, and it is much less permeable to water vapor and gastric fluids.

In some embodiments, the coating can, and usually does, contain aplasticizer and possibly other coating excipients such as colorants,talc, and/or magnesium stearate, which are well known in the art.Suitable plasticizers include triethyl citrate (Citroflex 2), triacetin(glyceryl triacetate), acetyl triethyl citrate (Citroflec A2), Carbowax400 (polyethylene glycol 400), diethyl phthalate, tributyl citrate,acetylated monoglycerides, glycerol, fatty acid esters, propyleneglycol, and dibutyl phthalate. In particular, anionic carboxylic acrylicpolymers usually will contain 10-25% by weight of a plasticizer,especially dibutyl phthalate, polyethylene glycol, triethyl citrate andtriacetin. Conventional coating techniques such as spray or pan coatingare employed to apply coatings. The coating thickness must be sufficientto ensure that the oral dosage form remains intact until the desiredsite of topical delivery in the intestinal tract is reached.

Colorants, detackifiers, surfactants, antifoaming agents, lubricants(e.g., carnuba wax or PEG) may be added to the coatings besidesplasticizers to solubilize or disperse the coating material, and toimprove coating performance and the coated product.

In other embodiments, the formulations described herein, which includeibrutinib and/or an anticancer agent, are delivered using a pulsatiledosage form. A pulsatile dosage form is capable of providing one or moreimmediate release pulses at predetermined time points after a controlledlag time or at specific sites. Many other types of controlled releasesystems known to those of ordinary skill in the art and are suitable foruse with the formulations described herein. Examples of such deliverysystems include, e.g., polymer-based systems, such as polylactic andpolyglycolic acid, plyanhydrides and polycaprolactone; porous matrices,nonpolymer-based systems that are lipids, including sterols, such ascholesterol, cholesterol esters and fatty acids, or neutral fats, suchas mono-, di- and triglycerides; hydrogel release systems; silasticsystems; peptide-based systems; wax coatings, bioerodible dosage forms,compressed tablets using conventional binders and the like. See, e.g.,Liberman et al., Pharmaceutical Dosage Forms, 2 Ed., Vol. 1, pp. 209-214(1990); Singh et al., Encyclopedia of Pharmaceutical Technology, 2ndEd., pp. 751-753 (2002); U.S. Pat. Nos. 4,327,725, 4,624,848, 4,968,509,5,461,140, 5,456,923, 5,516,527, 5,622,721, 5,686,105, 5,700,410,5,977,175, 6,465,014 and 6,932,983.

In some embodiments, pharmaceutical formulations are provided thatinclude particles of ibrutinib and/or an anticancer agent, describedherein and at least one dispersing agent or suspending agent for oraladministration to a subject. The formulations may be a powder and/orgranules for suspension, and upon admixture with water, a substantiallyuniform suspension is obtained.

Liquid formulation dosage forms for oral administration can be aqueoussuspensions selected from the group including, but not limited to,pharmaceutically acceptable aqueous oral dispersions, emulsions,solutions, elixirs, gels, and syrups. See, e.g., Singh et al.,Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 754-757 (2002).In addition the liquid dosage forms may include additives, such as: (a)disintegrating agents; (b) dispersing agents; (c) wetting agents; (d) atleast one preservative, (e) viscosity enhancing agents, (0 at least onesweetening agent, and (g) at least one flavoring agent. In someembodiments, the aqueous dispersions can further include a crystallineinhibitor.

The aqueous suspensions and dispersions described herein can remain in ahomogenous state, as defined in The USP Pharmacists' Pharmacopeia (2005edition, chapter 905), for at least 4 hours. The homogeneity should bedetermined by a sampling method consistent with regard to determininghomogeneity of the entire composition. In one embodiment, an aqueoussuspension can be re-suspended into a homogenous suspension by physicalagitation lasting less than 1 minute. In another embodiment, an aqueoussuspension can be re-suspended into a homogenous suspension by physicalagitation lasting less than 45 seconds. In yet another embodiment, anaqueous suspension can be re-suspended into a homogenous suspension byphysical agitation lasting less than 30 seconds. In still anotherembodiment, no agitation is necessary to maintain a homogeneous aqueousdispersion.

Examples of disintegrating agents for use in the aqueous suspensions anddispersions include, but are not limited to, a starch, e.g., a naturalstarch such as corn starch or potato starch, a pregelatinized starchsuch as National 1551 or Amijel®, or sodium starch glycolate such asPromogel® or Explotab®; a cellulose such as a wood product,methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel®PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, andSolka-Floc®, methylcellulose, croscarmellose, or a cross-linkedcellulose, such as cross-linked sodium carboxymethylcellulose(Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linkedcroscarmellose; a cross-linked starch such as sodium starch glycolate; across-linked polymer such as crospovidone; a cross-linkedpolyvinylpyrrolidone; alginate such as alginic acid or a salt of alginicacid such as sodium alginate; a clay such as Veegum® HV (magnesiumaluminum silicate); a gum such as agar, guar, locust bean, Karaya,pectin, or tragacanth; sodium starch glycolate; bentonite; a naturalsponge; a surfactant; a resin such as a cation-exchange resin; citruspulp; sodium lauryl sulfate; sodium lauryl sulfate in combinationstarch; and the like.

In some embodiments, the dispersing agents suitable for the aqueoussuspensions and dispersions described herein are known in the art andinclude, for example, hydrophilic polymers, electrolytes, Tween® 60 or80, PEG, polyvinylpyrrolidone (PVP; commercially known as Plasdone®),and the carbohydrate-based dispersing agents such as, for example,hydroxypropylcellulose and hydroxypropyl cellulose ethers (e.g., HPC,HPC-SL, and HPC-L), hydroxypropyl methylcellulose and hydroxypropylmethylcellulose ethers (e.g. HPMC K100, HPMC K4M, HPMC K15M, and HPMCK100M), carboxymethylcellulose sodium, methylcellulose,hydroxyethylcellulose, hydroxypropylmethyl-cellulose phthalate,hydroxypropylmethyl-cellulose acetate stearate, noncrystallinecellulose, magnesium aluminum silicate, triethanolamine, polyvinylalcohol (PVA), polyvinylpyrrolidone/vinyl acetate copolymer (Plasdone®,e.g., S-630), 4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethyleneoxide and formaldehyde (also known as tyloxapol), poloxamers (e.g.,Pluronics F68®, F88®, and F108®, which are block copolymers of ethyleneoxide and propylene oxide); and poloxamines (e.g., Tetronic 908®, alsoknown as Poloxamine 908®, which is a tetrafunctional block copolymerderived from sequential addition of propylene oxide and ethylene oxideto ethylenediamine (BASF Corporation, Parsippany, N.J.)). In otherembodiments, the dispersing agent is selected from a group notcomprising one of the following agents: hydrophilic polymers;electrolytes; Tween 60 or 80; PEG; polyvinylpyrrolidone (PVP);hydroxypropylcellulose and hydroxypropyl cellulose ethers (e.g., HPC,HPC-SL, and HPC-L); hydroxypropyl methylcellulose and hydroxypropylmethylcellulose ethers (e.g. HPMC K100, HPMC K4M, HPMC K15M, HPMC K100M,and Pharmacoat® USP 2910 (Shin-Etsu)); carboxymethylcellulose sodium;methylcellulose; hydroxyethylcellulose; hydroxypropylmethyl-cellulosephthalate; hydroxypropylmethyl-cellulose acetate stearate;non-crystalline cellulose; magnesium aluminum silicate; triethanolamine;polyvinyl alcohol (PVA); 4-(1,1,3,3-tetramethylbutyl)-phenol polymerwith ethylene oxide and formaldehyde; poloxamers (e.g., Pluronics F68®,F88®, and F108®, which are block copolymers of ethylene oxide andpropylene oxide); or poloxamines (e.g., Tetronic 908®, also known asPoloxamine 908®).

Wetting agents suitable for the aqueous suspensions and dispersionsdescribed herein are known in the art and include, but are not limitedto, cetyl alcohol, glycerol monostearate, polyoxyethylene sorbitan fattyacid esters (e.g., the commercially available Tweens® such as e.g.,Tween 20® and Tween 80® (ICI Specialty Chemicals)), and polyethyleneglycols (e.g., Carbowaxs 3350® and 1450®, and Carbopol 934® (UnionCarbide)), oleic acid, glyceryl monostearate, sorbitan monooleate,sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitanmonooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, sodiumlauryl sulfate, sodium docusate, triacetin, vitamin E TPGS, sodiumtaurocholate, simethicone, phosphotidylcholine and the like.

Suitable preservatives for the aqueous suspensions or dispersionsdescribed herein include, for example, potassium sorbate, parabens(e.g., methylparaben and propylparaben), benzoic acid and its salts,other esters of parahydroxybenzoic acid such as butylparaben, alcoholssuch as ethyl alcohol or benzyl alcohol, phenolic compounds such asphenol, or quaternary compounds such as benzalkonium chloride.Preservatives, as used herein, are incorporated into the dosage form ata concentration sufficient to inhibit microbial growth.

Suitable viscosity enhancing agents for the aqueous suspensions ordispersions described herein include, but are not limited to, methylcellulose, xanthan gum, carboxymethyl cellulose, hydroxypropylcellulose, hydroxypropylmethyl cellulose, Plasdon® S-630, carbomer,polyvinyl alcohol, alginates, acacia, chitosans and combinationsthereof. The concentration of the viscosity enhancing agent will dependupon the agent selected and the viscosity desired.

Examples of sweetening agents suitable for the aqueous suspensions ordispersions described herein include, for example, acacia syrup,acesulfame K, alitame, anise, apple, aspartame, banana, Bavarian cream,berry, black currant, butterscotch, calcium citrate, camphor, caramel,cherry, cherry cream, chocolate, cinnamon, bubble gum, citrus, citruspunch, citrus cream, cotton candy, cocoa, cola, cool cherry, coolcitrus, cyclamate, cylamate, dextrose, eucalyptus, eugenol, fructose,fruit punch, ginger, glycyrrhetinate, glycyrrhiza (licorice) syrup,grape, grapefruit, honey, isomalt, lemon, lime, lemon cream,monoammonium glyrrhizinate (MagnaSweet®), maltol, mannitol, maple,marshmallow, menthol, mint cream, mixed berry, neohesperidine DC,neotame, orange, pear, peach, peppermint, peppermint cream, Prosweet®Powder, raspberry, root beer, rum, saccharin, safrole, sorbitol,spearmint, spearmint cream, strawberry, strawberry cream, stevia,sucralose, sucrose, sodium saccharin, saccharin, aspartame, acesulfamepotassium, mannitol, talin, sucralose, sorbitol, swiss cream, tagatose,tangerine, thaumatin, tutti fruitti, vanilla, walnut, watermelon, wildcherry, wintergreen, xylitol, or any combination of these flavoringingredients, e.g., anise-menthol, cherry-anise, cinnamon-orange,cherry-cinnamon, chocolate-mint, honey-lemon, lemon-lime, lemon-mint,menthol-eucalyptus, orange-cream, vanilla-mint, and mixtures thereof. Inone embodiment, the aqueous liquid dispersion can comprise a sweeteningagent or flavoring agent in a concentration ranging from about 0.001% toabout 1.0% the volume of the aqueous dispersion. In another embodiment,the aqueous liquid dispersion can comprise a sweetening agent orflavoring agent in a concentration ranging from about 0.005% to about0.5% the volume of the aqueous dispersion. In yet another embodiment,the aqueous liquid dispersion can comprise a sweetening agent orflavoring agent in a concentration ranging from about 0.01% to about1.0% the volume of the aqueous dispersion.

In addition to the additives listed above, the liquid formulations canalso include inert diluents commonly used in the art, such as water orother solvents, solubilizing agents, and emulsifiers. Exemplaryemulsifiers are ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propyleneglycol,1,3-butyleneglycol, dimethylformamide, sodium lauryl sulfate, sodiumdoccusate, cholesterol, cholesterol esters, taurocholic acid,phosphotidylcholine, oils, such as cottonseed oil, groundnut oil, corngerm oil, olive oil, castor oil, and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters ofsorbitan, or mixtures of these substances, and the like.

In some embodiments, the pharmaceutical formulations described hereincan be self-emulsifying drug delivery systems (SEDDS). Emulsions aredispersions of one immiscible phase in another, usually in the form ofdroplets. Generally, emulsions are created by vigorous mechanicaldispersion. SEDDS, as opposed to emulsions or microemulsions,spontaneously form emulsions when added to an excess of water withoutany external mechanical dispersion or agitation. An advantage of SEDDSis that only gentle mixing is required to distribute the dropletsthroughout the solution. Additionally, water or the aqueous phase can beadded just prior to administration, which ensures stability of anunstable or hydrophobic active ingredient. Thus, the SEDDS provides aneffective delivery system for oral and parenteral delivery ofhydrophobic active ingredients. SEDDS may provide improvements in thebioavailability of hydrophobic active ingredients. Methods of producingself-emulsifying dosage forms are known in the art and include, but arenot limited to, for example, U.S. Pat. Nos. 5,858,401, 6,667,048, and6,960,563, each of which is specifically incorporated by reference.

It is to be appreciated that there is overlap between the above-listedadditives used in the aqueous dispersions or suspensions describedherein, since a given additive is often classified differently bydifferent practitioners in the field, or is commonly used for any ofseveral different functions. Thus, the above-listed additives should betaken as merely exemplary, and not limiting, of the types of additivesthat can be included in formulations described herein. The amounts ofsuch additives can be readily determined by one skilled in the art,according to the particular properties desired.

Intranasal Formulations

Intranasal formulations are known in the art and are described in, forexample, U.S. Pat. Nos. 4,476,116, 5,116,817 and 6,391,452, each ofwhich is specifically incorporated by reference. Formulations thatinclude ibrutinib and/or An anticancer agent, which are preparedaccording to these and other techniques well-known in the art areprepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, fluorocarbons, and/or other solubilizing ordispersing agents known in the art. See, for example, Ansel, H. C. etal., Pharmaceutical Dosage Forms and Drug Delivery Systems, Sixth Ed.(1995). Preferably these compositions and formulations are prepared withsuitable nontoxic pharmaceutically acceptable ingredients. Theseingredients are known to those skilled in the preparation of nasaldosage forms and some of these can be found in REMINGTON: THE SCIENCEAND PRACTICE OF PHARMACY, 21st edition, 2005, a standard reference inthe field. The choice of suitable carriers is highly dependent upon theexact nature of the nasal dosage form desired, e.g., solutions,suspensions, ointments, or gels. Nasal dosage forms generally containlarge amounts of water in addition to the active ingredient. Minoramounts of other ingredients such as pH adjusters, emulsifiers ordispersing agents, preservatives, surfactants, gelling agents, orbuffering and other stabilizing and solubilizing agents may also bepresent. The nasal dosage form should be isotonic with nasal secretions.

For administration by inhalation described herein may be in a form as anaerosol, a mist or a powder. Pharmaceutical compositions describedherein are conveniently delivered in the form of an aerosol spraypresentation from pressurized packs or a nebulizer, with the use of asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, such as, by way of example only, gelatin foruse in an inhaler or insufflator may be formulated containing a powdermix of the compound described herein and a suitable powder base such aslactose or starch.

Buccal Formulations

Buccal formulations may be administered using a variety of formulationsknown in the art. For example, such formulations include, but are notlimited to, U.S. Pat. Nos. 4,229,447, 4,596,795, 4,755,386, and5,739,136, each of which is specifically incorporated by reference. Inaddition, the buccal dosage forms described herein can further include abioerodible (hydrolysable) polymeric carrier that also serves to adherethe dosage form to the buccal mucosa. The buccal dosage form isfabricated so as to erode gradually over a predetermined time period,wherein the delivery is provided essentially throughout. Buccal drugdelivery, as will be appreciated by those skilled in the art, avoids thedisadvantages encountered with oral drug administration, e.g., slowabsorption, degradation of the active agent by fluids present in thegastrointestinal tract and/or first-pass inactivation in the liver. Withregard to the bioerodible (hydrolysable) polymeric carrier, it will beappreciated that virtually any such carrier can be used, so long as thedesired drug release profile is not compromised, and the carrier iscompatible with ibrutinib and/or An anticancer agent, and any othercomponents that may be present in the buccal dosage unit. Generally, thepolymeric carrier comprises hydrophilic (water-soluble andwater-swellable) polymers that adhere to the wet surface of the buccalmucosa. Examples of polymeric carriers useful herein include acrylicacid polymers and co, e.g., those known as “carbomers” (Carbopol®, whichmay be obtained from B.F. Goodrich, is one such polymer). Othercomponents may also be incorporated into the buccal dosage formsdescribed herein include, but are not limited to, disintegrants,diluents, binders, lubricants, flavoring, colorants, preservatives, andthe like. For buccal or sublingual administration, the compositions maytake the form of tablets, lozenges, or gels formulated in a conventionalmanner.

Transdermal Formulations

Transdermal formulations described herein may be administered using avariety of devices which have been described in the art. For example,such devices include, but are not limited to, U.S. Pat. Nos. 3,598,122,3,598,123, 3,710,795, 3,731,683, 3,742,951, 3,814,097, 3,921,636,3,972,995, 3,993,072, 3,993,073, 3,996,934, 4,031,894, 4,060,084,4,069,307, 4,077,407, 4,201,211, 4,230,105, 4,292,299, 4,292,303,5,336,168, 5,665,378, 5,837,280, 5,869,090, 6,923,983, 6,929,801 and6,946,144, each of which is specifically incorporated by reference inits entirety.

The transdermal dosage forms described herein may incorporate certainpharmaceutically acceptable excipients which are conventional in theart. In one embodiments, the transdermal formulations described hereininclude at least three components: (1) a formulation of a compound ofibrutinib and An anticancer agent; (2) a penetration enhancer; and (3)an aqueous adjuvant. In addition, transdermal formulations can includeadditional components such as, but not limited to, gelling agents,creams and ointment bases, and the like. In some embodiments, thetransdermal formulation can further include a woven or non-woven backingmaterial to enhance absorption and prevent the removal of thetransdermal formulation from the skin. In other embodiments, thetransdermal formulations described herein can maintain a saturated orsupersaturated state to promote diffusion into the skin.

Formulations suitable for transdermal administration of compoundsdescribed herein may employ transdermal delivery devices and transdermaldelivery patches and can be lipophilic emulsions or buffered, aqueoussolutions, dissolved and/or dispersed in a polymer or an adhesive. Suchpatches may be constructed for continuous, pulsatile, or on demanddelivery of pharmaceutical agents. Still further, transdermal deliveryof the compounds described herein can be accomplished by means ofiontophoretic patches and the like. Additionally, transdermal patchescan provide controlled delivery of ibrutinib and An anticancer agent.The rate of absorption can be slowed by using rate-controlling membranesor by trapping the compound within a polymer matrix or gel. Conversely,absorption enhancers can be used to increase absorption. An absorptionenhancer or carrier can include absorbable pharmaceutically acceptablesolvents to assist passage through the skin. For example, transdermaldevices are in the form of a bandage comprising a backing member, areservoir containing the compound optionally with carriers, optionally arate controlling barrier to deliver the compound to the skin of the hostat a controlled and predetermined rate over a prolonged period of time,and means to secure the device to the skin.

Injectable Formulations

Formulations that include a compound of ibrutinib and/or An anticanceragent, suitable for intramuscular, subcutaneous, or intravenousinjection may include physiologically acceptable sterile aqueous ornon-aqueous solutions, dispersions, suspensions or emulsions, andsterile powders for reconstitution into sterile injectable solutions ordispersions. Examples of suitable aqueous and non-aqueous carriers,diluents, solvents, or vehicles including water, ethanol, polyols(propyleneglycol, polyethylene-glycol, glycerol, cremophor and thelike), suitable mixtures thereof, vegetable oils (such as olive oil) andinjectable organic esters such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case ofdispersions, and by the use of surfactants. Formulations suitable forsubcutaneous injection may also contain additives such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the growth ofmicroorganisms can be ensured by various antibacterial and antifungalagents, such as parabens, chlorobutanol, phenol, sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, such as aluminum monostearate and gelatin.

For intravenous injections, compounds described herein may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological salinebuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art. For other parenteral injections, appropriateformulations may include aqueous or nonaqueous solutions, preferablywith physiologically compatible buffers or excipients. Such excipientsare generally known in the art.

Parenteral injections may involve bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The pharmaceutical composition described herein may be ina form suitable for parenteral injection as a sterile suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Pharmaceutical formulations for parenteral administrationinclude aqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

Other Formulations

In certain embodiments, delivery systems for pharmaceutical compoundsmay be employed, such as, for example, liposomes and emulsions. Incertain embodiments, compositions provided herein can also include anmucoadhesive polymer, selected from among, for example,carboxymethylcellulose, carbomer (acrylic acid polymer),poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylicacid/butyl acrylate copolymer, sodium alginate and dextran.

In some embodiments, the compounds described herein may be administeredtopically and can be formulated into a variety of topicallyadministrable compositions, such as solutions, suspensions, lotions,gels, pastes, medicated sticks, balms, creams or ointments. Suchpharmaceutical compounds can contain solubilizers, stabilizers, tonicityenhancing agents, buffers and preservatives.

The compounds described herein may also be formulated in rectalcompositions such as enemas, rectal gels, rectal foams, rectal aerosols,suppositories, jelly suppositories, or retention enemas, containingconventional suppository bases such as cocoa butter or other glycerides,as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and thelike. In suppository forms of the compositions, a low-melting wax suchas, but not limited to, a mixture of fatty acid glycerides, optionallyin combination with cocoa butter is first melted.

Dosing and Treatment Regimens

In some embodiments, the amount of ibrutinib that is administered incombination with an anticancer agent is from 10 mg/day up to, andincluding, 1000 mg/day. In some embodiments, the amount of ibrutinibthat is administered is from about 40 mg/day to 70 mg/day. In someembodiments, the amount of ibrutinib that is administered per day isabout 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg,about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg,about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about110 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, or about140 mg. In some embodiments, the amount of brutinib that is administeredis about 40 mg/day. In some embodiments, the amount of ibrutinib that isadministered is about 50 mg/day. In some embodiments, the amount ofibrutinib that is administered is about 60 mg/day. In some embodiments,the amount of ibrutinib that is administered is about 70 mg/day. In someembodiments, the amount of ibrutinib that is administered per day isabout 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg,about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg,about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg,about 500 mg, about 520 mg, about 540 mg, about 560 mg, about 580 mg, orabout 600 mg. In some embodiments, the amount of ibrutinib that isadministered per day is less than about 10 mg, or greater than about1000 mg.

In some embodiments, the amount of an anticancer agent that isadministered in combination with ibrutinib is from 0.01 μM to, andincluding, 100 μM. In some embodiments, the amount of an anticanceragent is from about 0.01 μM to about 100 μM. In some embodiments, theanticancer agent is an inhibitor of MALT1, JAK3, MCL-1 or IDH1. In someembodiments, the amount of a MALT1 inhibitor that is administered incombination with ibrutinib is from 0.01 μM to, and including, 100 μM. Insome embodiments, the amount of a MALT1 inhibitor is from about 0.01 μMto about 100 μM. In some embodiments, the amount of a JAK3 inhibitorthat is administered in combination with ibrutinib is from 0.01 μM to,and including, 100 μM. In some embodiments, the amount of a JAK3inhibitor is from about 0.01 μM to about 100 μM. In some embodiments,the amount of a MCL-1 inhibitor that is administered in combination withibrutinib is from 0.01 μM to, and including, 100 μM. In someembodiments, the amount of a MCL-1 inhibitor is from about 0.01 μM toabout 100 μM. In some embodiments, the amount of an IDH1 inhibitor thatis administered in combination with ibrutinib is from 0.01 μM to, andincluding, 100 μM. In some embodiments, the amount of an IDH1 inhibitoris from about 0.01 μM to about 100 μM.

In some embodiments, the amount of PIM inhibitor that is administered incombination with ibrutinib is from 0.01 μM to, and including, 100 μM. Insome embodiments, the amount of PIM inhibitor is from about 0.01 μM toabout 100 μM.

In some embodiments, ibrutinib is administered once per day, twice perday, or three times per day. In some embodiments, ibrutinib isadministered once per day. In some embodiments, an anticancer agent isadministered once per day, twice per day, or three times per day. Insome embodiments, an anticancer agent is administered once per day. Insome embodiments, ibrutinib and an anticancer agent are co-administered(e.g., in a single dosage form), once per day. In some embodiments, theanticancer agent is an inhibitor of MALT1, JAK3, MCL-1 or IDH1. In someembodiments, ibrutinib and a MALT1 inhibitor are co-administered (e.g.,in a single dosage form), once per day. In some embodiments, ibrutiniband a JAK3 inhibitor are co-administered (e.g., in a single dosageform), once per day. In some embodiments, ibrutinib and a MCL-1inhibitor are co-administered (e.g., in a single dosage form), once perday. In some embodiments, ibrutinib and an IDH1 inhibitor areco-administered (e.g., in a single dosage form), once per day. In someembodiments, the PIM inhibitor is administered once per day, twice perday, or three times per day. In some embodiments, ibrutinib and the PIMinhibitor are co-administered (e.g., in a single dosage form), once perday.

In some embodiments, the compositions disclosed herein are administeredfor prophylactic, therapeutic, or maintenance treatment. In someembodiments, the compositions disclosed herein are administered fortherapeutic applications. In some embodiments, the compositionsdisclosed herein are administered for therapeutic applications. In someembodiments, the compositions disclosed herein are administered as amaintenance therapy, for example for a patient in remission.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the compounds may be givencontinuously; alternatively, the dose of drug being administered may betemporarily reduced or temporarily suspended for a certain length oftime (i.e., a “drug holiday”). The length of the drug holiday can varybetween 2 days and 1 year, including by way of example only, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days,180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or365 days. The dose reduction during a drug holiday may be from 10%-100%,including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be reduced, as a function ofthe symptoms, to a level at which the improved disease, disorder orcondition is retained. Patients can, however, require intermittenttreatment on a long-term basis upon any recurrence of symptoms.

The amount of a given agent that will correspond to such an amount willvary depending upon factors such as the particular compound, theseverity of the disease, the identity (e.g., weight) of the subject orhost in need of treatment, but can nevertheless be routinely determinedin a manner known in the art according to the particular circumstancessurrounding the case, including, e.g., the specific agent beingadministered, the route of administration, and the subject or host beingtreated. In general, however, doses employed for adult human treatmentwill typically be in the range of 0.02-5000 mg per day, or from about1-1500 mg per day. The desired dose may conveniently be presented in asingle dose or as divided doses administered simultaneously (or over ashort period of time) or at appropriate intervals, for example as two,three, four or more sub-doses per day.

The pharmaceutical composition described herein may be in unit dosageforms suitable for single administration of precise dosages. In unitdosage form, the formulation is divided into unit doses containingappropriate quantities of one or more compound. The unit dosage may bein the form of a package containing discrete quantities of theformulation. Non-limiting examples are packaged tablets or capsules, andpowders in vials or ampoules. Aqueous suspension compositions can bepackaged in single-dose non-reclosable containers. Alternatively,multiple-dose reclosable containers can be used, in which case it istypical to include a preservative in the composition. By way of exampleonly, formulations for parenteral injection may be presented in unitdosage form, which include, but are not limited to ampoules, or inmulti-dose containers, with an added preservative.

The foregoing ranges are merely suggestive, as the number of variablesin regard to an individual treatment regime is large, and considerableexcursions from these recommended values are not uncommon. Such dosagesmay be altered depending on a number of variables, not limited to theactivity of the compound used, the disease or condition to be treated,the mode of administration, the requirements of the individual subject,the severity of the disease or condition being treated, and the judgmentof the practitioner.

Toxicity and therapeutic efficacy of such therapeutic regimens can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, including, but not limited to, the determinationof the LD50 (the dose lethal to 50% of the population) and the ED50 (thedose therapeutically effective in 50% of the population). The dose ratiobetween the toxic and therapeutic effects is the therapeutic index andit can be expressed as the ratio between LD50 and ED50. Compoundsexhibiting high therapeutic indices are preferred. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED50 with minimal toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized.

Kits/Article of Manufacture

Disclosed herein, in certain embodiments, are kits and articles ofmanufacture for use with one or more methods described herein. Such kitsinclude a carrier, package, or container that is compartmentalized toreceive one or more containers such as vials, tubes, and the like, eachof the container(s) comprising one of the separate elements to be usedin a method described herein. Suitable containers include, for example,bottles, vials, syringes, and test tubes. In one embodiment, thecontainers are formed from a variety of materials such as glass orplastic.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, bags, containers, bottles,and any packaging material suitable for a selected formulation andintended mode of administration and treatment.

For example, the container(s) include ibrutinib, optionally in acomposition or in combination with an anticancer agent that inhibitsIDH1, MCL-1 or MALT1 as disclosed herein. In some embodiments, thecontainer may include a PIM inhibitor. Such kits optionally include anidentifying description or label or instructions relating to its use inthe methods described herein.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

In one embodiment, a label is on or associated with the container. Inone embodiment, a label is on a container when letters, numbers or othercharacters forming the label are attached, molded or etched into thecontainer itself; a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In one embodiment, a label is used toindicate that the contents are to be used for a specific therapeuticapplication. The label also indicates directions for use of thecontents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented ina pack or dispenser device which contains one or more unit dosage formscontaining a compound provided herein. The pack, for example, containsmetal or plastic foil, such as a blister pack. In one embodiment, thepack or dispenser device is accompanied by instructions foradministration. In one embodiment, the pack or dispenser is alsoaccompanied with a notice associated with the container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the drug for human or veterinary administration.Such notice, for example, is the labeling approved by the U.S. Food andDrug Administration for prescription drugs, or the approved productinsert. In one embodiment, compositions containing a compound providedherein formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1: Combined Drug Treatment for Cell Viability Using Ibrutinib inCombination with IDH1 or MALT1 Inhibitors

Different DLBCL cell lines were tested in vitro to determine thesynergistic and antagonistic effect of ibrutinib with either IDH1 orMALT1 inhibitors.

The DLBCL cell lines used during the experiments included TMD8, OCI-LY3,OCI-LY10, U-2932 and SU-DHL-2.

DLBCL cells at either 1×10⁴ cells or 2×10⁴ cells were plated onto eachwell of a 96-W plate (Table 1).

TABLE 1 Cells Medium cells/well (200 ul) cells/ml TMD8 R-10 + S 1000050000 OCI-LY-3 IM-10 10000 50000 OCI-LY-10 IM-10 10000 50000 U-2932 R-1020000 100000 SU-DHL-2 R-10 20000 100000

Ibrutinib at 10000, 2000, 400, 80, 16, 3.2, 0.64, 0.128, 0.0256, and 0nM concentrations were used during the experiments. The concentrationsof the IDH1 inhibitor AGI5198 and the MALT1 inhibitor MI-2 are shown inTable 2. The stock solution for ibrutinib was prepared at 20 mMconcentration. The stock solutions for the IDH1 inhibitor AGI5198 andthe MALT1 inhibitor MI-2 were each prepared at 50 mM concentration.

TABLE 2 TMD8 LY-3 LY-10 U-2932 SU-DHL-2 AGI5198  10 uM 10 uM 10 uM  10uM  10 uM MI-2 100 nM  5 uM  5 uM 200 nM 200 nM

To each well of a 96-W plate was added 100 μL ibrutinib (2× of targetconcentration; diluted using appropriate cell medium for each cellline), 50 μL AGI5198 (IDH1 inhibitor) and/or MI-2 (MALT1 inhibitor) at4× of the target concentration, and 50 μL of cells (also at 4× targetconcentration). The 96-W plate was then incubated for 3 days. Cellviability was examined using a CellTiter-Glo assay.

CellTiter-Glo Assay

A 40 μL of CellTiter-Glo reagent was added directly into each well ofthe 96-W plate. The plate was then shaken on a Shaker (LabsystemWellmix) at speed 5 for 10-20 min at room temperature. Next, 100 μL ofthe mixed medium was transferred to a white, non-transparent, flatbottom 96-W plate for assaying. A Flexstation 3 luminometer was used fordetecting and measuring the luminescent signals. Measurements were takenat room temperature.

CellTiter-Glow reagents were thawed prior to use. Cells pre-plated ontoa second 96-W plate and incubated at room temperature for 30 minuteswere used for calibration purposes.

Table 3 indicates the experimental design layout on the 96-W plate.

TABLE 3 1 2 3 4 5 6 7 8 9 10 11 12

IDHi MALT1i Medium alone

indicates data missing or illegible when filed

Tables 4-8 illustrate the luminiscence for each cell line.

TABLE 4 TMD8 2 3 4 5 6 7 8 9 10 11 9604.64 19649.45 19936.75 22451.2422519.77 23004.75 22928.31 25788.09 25500.80 75993.70 IDH1i 9754.8821278.34 21591.99 22045.34 22888.78 24014.24 25405.91 23958.89 26784.4071955.73 MALT1i 10819.72 23421.20 25674.76 28263.05 27190.31 31383.7729180.29 32828.16 32496.06 84399.08 Medium alone 10000 2000 400 80 163.2 0.64 0.128 0.0256 0 ibrutinib (nM)

TABLE 5 OCI-LY-10 2 3 4 5 6 7 8 9 10 11 21999.40 22738.32 22543.7321796.92 22951.32 37017.16 49163.38 45182.13 60402.37 54469.95 IDH1i9734.85 10110.89 9064.30 9592.85 7749.49 13413.69 16971.57 17802.5321817.96 19317.19 MALT1i 24826.24 24452.84 23784.91 23511.43 27324.3851190.81 63983.91 68196.56 61548.88 58403.86 Medium alone 10000 2000 40080 16 3.2 0.64 0.128 0.0256 0 ibrutinib (nM)

TABLE 6 OCI-LY-3 2 3 4 5 6 7 8 9 10 11 58163.88 66282.58 68413.2865372.05 63970.82 61774.53 64267.34 58171.75 60845.63 61813.89 IDH1i23784.06 26843.66 25227.27 27617.74 27654.48 26610.12 24778.56 26368.7125371.59 23290.74 MALT1i 63477.51 55749.79 64472.02 68972.20 64655.7066789.02 63322.70 59441.78 63957.70 52879.12 Medium alone 10000 2000 40080 16 3.2 0.64 0.128 0.0256 0 ibrutinib (nM)

TABLE 7 U2932 2 3 4 5 6 7 8 9 10 11 31970.32 43047.82 45769.76 43730.2843920.00 46620.86 45155.81 48958.10 47490.41 52209.68 IDH1i 31838.5745695.98 41263.93 47234.82 44565.57 48821.08 49419.22 46995.03 48183.4154720.82 MALT1i 37935.94 46858.01 46009.54 49777.58 51023.93 53031.7953722.16 57150.28 63123.80 65814.13 Medium alone 10000 2000 400 80 163.2 0.64 0.128 0.0256 0 ibrutinib (nM)

TABLE 8 SU-DHL-2 2 3 4 5 6 7 8 9 10 11 50666.3 149634.6 145121.4152613.5 152240.5 156751.0 142615.3 145229.1 130906.9 124126.7 IDH1i61725.8 151552.3 152400.8 154662.6 153735.3 156036.5 148058.4 145168.7127667.9 131429.7 MALT1i 111485.8 154426.1 159908.6 164140.7 155353.5161613.5 159399.0 146159.1 141202.0 143348.2 Medium alone 10000 2000 40080 16 3.2 0.64 0.128 0.0256 0 ibrutinib (nM)

The luminescent measurements were subsequently process and analyzed toderive combination index (CI) for either the combination of ibrutiniband the IDH1 inhibitor AGI5198, or the combination of ibrutinib andMALT1 inhibitor MI-2 at each cell line. CI is a quantitative descriptionof the interaction property of the combination of two drugs. In general,the combination is described as synergistic (CI<1), additive (CI=1), orantagonistic (CI>1). Synergism is further separated into very strongsynergism (<0.1), strong synergism (0.1-0.3), synergism (0.3-0.7),moderate synergism (0.7-0.85), and slight synergism (0.85-0.9). Tables9-23 illustrate the CI values for the combinations of ibrutinib witheither the IDH1 inhibitor AGI5198 or with the MALT1 inhibitor MI-2 ineach cell line. Tables 9 and 10 illustrate the CI values for ibrutinibin combination with either the IDH1 inhibitor AGI5198 or with the MALT1inhibitor MI-2 in TMD8 cell line. Tables 11 and 12 illustrate the CIvalues for ibrutinib in combination with either the IDH1 inhibitorAGI5198 or with the MALT1 inhibitor MI-2 in OCI-LY10 cell line. Tables13 and 14 illustrate the CI values for ibrutinib in combination witheither the IDH1 inhibitor AGI5198 or with the MALT1 inhibitor MI-2 inOCI-LY3 cell line. Tables 15 and 16 illustrate the CI values foribrutinib in combination with either the IDH1 inhibitor AGI5198 or withthe MALT1 inhibitor MI-2 in U2932 cell line. Tables 17 and 18 illustratethe CI values for ibrutinib in combination with either the IDH1inhibitor AGI5198 or with the MALT1 inhibitor MI-2 in SU-DHL-2 cellline. The gray regions in Tables 9-14 indicate synergism for therespective ibrutinib and either IDH1 or MALT1 inhibitor combinations.

TABLE 9 ibrutinib + IDH1 Combination Ibrutinib AGI5198 CI 0.0256 100000.001 0.128 10000 0.006 0.64 10000 0.004 3.2 10000 0.022 16 10000 0.07680 10000 0.362 400 10000 0.257 2000 10000 1.02 10000 10000 0

TABLE 10 ibrutinib + MALT1 Combination Ibrutinib M1-2 CI 0.0256 1000.334 0.128 100 0.294 0.64 100 0.336 3.2 100 0.339 16 100 0.379 80 1000.534 400 100 1.207 2000 100 3.972 10000 100 0.123

TABLE 11 ibrutinib + IDH1 Combination Ibrutinib AGI5198 CI 0.0256 100002.131 0.128 10000 0.013 0.64 10000 0.132 3.2 10000 0.044 16 10000 0.01380 10000 0.049 400 10000 0.288 2000 10000 1.503 10000 10000 6.37

TABLE 12 ibrutinib + MALT1 Combination Ibrutinib M1-2 CI 0.0256 50001.367 0.128 5000 1.21 0.64 5000 1.178 3.2 5000 1.036 16 5000 0.787 805000 0.875 400 5000 0.855 2000 5000 0.94 10000 5000 1.067

TABLE 13 ibrutinib + IDH1 Combination Ibrutinib AGI5198 CI 0.0256 100000.463 0.128 10000 0.254 0.64 10000 1.591 3.2 10000 0.595 16 10000 3.13980 10000 1805.322 400 10000 2.24E+16 2000 10000 4.34E+06 10000 100000.256

TABLE 14 ibrutinib + MALT1 Combination Ibrutinib M1-2 CI 0.0256 50000.76 0.128 5000 0.829 0.64 5000 0.721 3.2 5000 0.846 16 5000 0.925 805000 0.922 400 5000 0.75 2000 5000 0.863 10000 5000 0.66

TABLE 15 ibrutinib + IDH1 Combination Ibrutinib AGI5198 CI 0.0256 100005.31 0.128 10000 6.927 0.64 10000 3.57 3.2 10000 4.575 16 10000 2.937 8010000 2.909 400 10000 4.84 2000 10000 3.959 10000 10000 0.609

TABLE 16 ibrutinib + MALT1 Combination Ibrutinib MI-2 CI 0.0256 2002.684 0.128 200 2.28 0.64 200 3.202 3.2 200 2.949 16 200 1.669 80 2002.519 400 200 1.18 2000 200 4.1 10000 200 0.432

TABLE 17 ibrutinib + IDH1 Combination Ibrutinib AGI5198 CI 0.0256 100000.307 0.128 10000 18.972 0.64 10000 7.625 3.2 10000 1.01E+04 16 10000443.373 80 10000 547.987 400 10000 18.37 2000 10000 118.285 10000 100000.142

TABLE 18 ibrutinib + MALT1 Combination Ibrutinib MI-2 CI 0.0256 2002.714 0.128 200 3.93 0.64 200 4.29 3.2 200 6.085 16 200 5.377 80 2005.687 400 200 5.292 2000 200 5.995 10000 200 1.379

Example 2: Combined Drug Treatment for Cell Viability Using Ibrutinib inCombination with JAK3 or MCL-1 Inhibitors

Different DLBCL cell lines were tested in vitro to determine thesynergistic and antagonistic effect of ibrutinib with MCL-1 or JAK3inhibitors.

The DLBCL cell lines used during the experiments included TMD8, OCI-LY3,OCI-LY10, U-2932, SU-DHL-2, and HBL-1.

DLBCL cells at either 1×10⁴ cells or 2×10⁴ cells were plated onto eachwell of a 96-W plate (Table 19).

TABLE 19 Cells cells/well (200 ul) cells/ml TMD8 10000 50000 OCI-LY-310000 50000 OCI-LY-10 10000 50000 HBL-1 10000 50000 U-2932 20000 100000SU-DHL-2 20000 100000

The culture medium used for TMD8 cells was R-10+S, for OCI-LY-3 andOCI-LY-10 cells were IM-10, and for U-2932 and SU-DHL-2 cells were R-10.

Ibrutinib at 20000, 4000, 800, 160, 32, 6.4, 1.28, 0.256, 0.0512, and 0nM concentrations were used during the experiments. The concentrationsof the JAK3 and MCL-1 inhibitors are shown in Table 20.

TABLE 20 TMD8 OCI-LY-10 OCI-LY-3 HBL-1 U-2932 SU-DHL-2 JAK3 10 μM 10 μM10 μM 10 μM 10 μM 10 μM MCL-1  3 μM  3 μM  3 μM  3 μM  3 μM  3 μM

To each well of a 96-W plate was added ibrutinib at a finalconcentration of 20000, 4000, 800, 160, 32, 6.4, 1.28, 0.256, 0.0512, or0 nM, either JAK3 inhibitor tofacitinib at final concentration of 10 μM,or MCL-1 inhibitor MIM1 at a final concentration of 3 μM, andappropriate concentration of cells to achieve a concentration of either1×10⁴ cells or 2×10⁴ cells per well. The 96-W plate was then incubatedfor 3 days. Cell viability was examined using a CellTiter-Glo assay.

CellTiter-Glo Assay

A 40 μL of CellTiter-Glo reagent was added directly into each well ofthe 96-W plate. The plate was then shaken on a Shaker (LabsystemWellmix) at speed 5 for 10-20 min at room temperature. Next, 100 μL ofthe mixed medium was transferred to a white, non-transparent, flatbottom 96-W plate for assaying. A Flexstation 3 luminometer was used fordetecting and measuring the luminescent signals. Measurements were takenat room temperature.

CellTiter-Glow reagents were thawed prior to use. Cells pre-plated ontoa second 96-W plate and incubated at room temperature for 30 minuteswere used for calibration purposes.

Table 21 indicates the experimental design layout on the 96-W plate.

TABLE 21 1 2 3 4 5 6 7 8 9 10 11 12

JAK3

MCL-1

Medium alone

indicates data missing or illegible when filed

Tables 22-27 illustrate the luminiscence for each cell line.

TABLE 22 TMD8 2 3 4 5 6 7 8 9 10 11 6463.53 19962.15 21620.76 23674.6723770.82 22806.63 23722.74 22844.02 22758.56 42085.03 JAK3 4767.5215624.64 18063.15 19452.01 19032.68 19112.81 18591.98 18570.62 19059.3936019.47 MCL-1 5261.63 22488.80 25963.61 29096.55 28802.75 28578.4028727.97 28516.97 28324.66 60810.56 Medium Alone 20000 40000 800 160 326.4 1.28 0.256 0.0512 0 ibrutinib (nM)

TABLE 23 OCI-LY-10 2 3 4 5 6 7 8 9 10 11 14383.00 14751.73 14159.6113583.65 13432.93 19547.86 35330.40 42984.83 51855.78 55820.26 JAK39228.91 10886.83 9390.40 8938.24 8025.84 11024.10 18751.19 26007.2933524.45 43143.62 MCL-1 14261.89 15817.53 14708.66 13712.84 14003.5117144.41 28222.33 40290.71 46968.14 48042.02 Medium Alone 20000 40000800 160 32 6.4 1.28 0.256 0.0512 0 ibrutinib (nM)

TABLE 24 OCI-LY-3 2 3 4 5 6 7 8 9 10 11 59782.00 45217.97 54193.4442659.67 47894.25 52737.31 49371.84 56590.84 52005.22 60068.93 JAK375182.71 74901.14 74844.82 65609.22 75378.47 73884.80 80436.06 80052.5976311.68 80237.62 MCL-1 69084.64 75112.98 82227.41 84190.37 80596.9681709.84 89730.66 81910.97 80095.49 67896.67 Medium Alone 20000 40000800 160 32 6.4 1.28 0.256 0.0512 0 ibrutinib (nM)

TABLE 25 HBL-1 2 3 4 5 6 7 8 9 10 11 17614.87 39380.73 37551.94 41745.8247226.84 46403.61 56756.95 49532.94 48007.16 78088.40 JAK3 7366.1226941.18 28815.56 32832.47 31548.03 30743.57 35637.34 29488.62 27898.4851241.07 MCL-1 16831.87 36806.47 35103.71 35090.31 35516.66 38857.8343461.99 42826.48 46288.31 89720.81 Medium Alone 20000 40000 800 160 326.4 1.28 0.256 0.0512 0 ibrutinib (nM)

TABLE 26 U2932 2 3 4 5 6 7 8 9 10 11 24327.81 54924.70 54213.58 55595.7257921.57 58424.16 61987.79 61314.09 61632.23 74370.91 JAK3 17553.4549251.78 51419.90 50938.68 51898.43 53424.93 54122.68 53088.09 55451.3659686.00 MCL-1 18900.83 55951.28 54579.83 55090.45 57009.94 58640.7160811.50 60528.12 62396.81 61068.14 Medium Alone 20000 40000 800 160 326.4 1.28 0.256 0.0512 0 ibrutinib (nM)

TABLE 27 SU-DHL-2 2 3 4 5 6 7 8 9 10 11 2706.8 164389.1 167444.0165414.5 173773.3 165146.8 171896.5 167037.0 154568.4 133588.4 JAK317553.4 49251.8 51419.9 50938.7 51898.4 53424.9 54122.7 53088.1 55451.459686.0 MCL-1 4650.6 180616.8 181165.6 183505.7 180464.1 178437.4185508.3 180865.8 168606.0 153374.3 Medium Alone 20000 40000 800 160 326.4 1.28 0.256 0.0512 0 ibrutinib (nM)

The luminescent measurements were subsequently processed and analyzed toderive combination index (CI) for combination of ibrutinib with eitherthe JAK3 inhibitor tofacitinib, or the MCL-1 inhibitor MIM1 at each cellline. CI is a quantitative description of the interaction property ofthe combination of two drugs. In general, the combination is describedas synergistic (CI<1), additive (CI=1), or antagonistic (CI>1).Synergism is further separated into very strong synergism (<0.1), strongsynergism (0.1-0.3), synergism (0.3-0.7), moderate synergism (0.7-0.85),and slight synergism (0.85-0.9). Tables 28-39 illustrate the CI valuesfor each ibrutinib and JAK3 or MCL-1 inhibitor combination in each cellline. Tables 28 and 29 illustrate the CI values for ibrutinib incombination with either the JAK3 inhibitor tofacitinib or the MCL-1inhibitor MIM1 in TMD8 cell line. Tables 30 and 31 illustrate the CIvalues for ibrutinib in combination with either the JAK3 inhibitortofacitinib or the MCL-1 inhibitor MIM1 in OCI-LY-10 cell line. Tables32 and 33 illustrate the CI values for ibrutinib in combination witheither the JAK3 inhibitor tofacitinib or the MCL-1 inhibitor MIM1 inOCI-LY-3 cell line. Tables 34 and 35 illustrate the CI values foribrutinib in combination with either the JAK3 inhibitor tofacitinib orthe MCL-1 inhibitor MIM1 in HBL-1 cell line. Tables 36 and 37 illustratethe CI values for ibrutinib in combination with either the JAK3inhibitor tofacitinib or the MCL-1 inhibitor MIM1 in U2932 cell line.Tables 38 and 39 illustrate the CI values for ibrutinib in combinationwith either the JAK3 inhibitor tofacitinib or the MCL-1 inhibitor MIM1in SU-DHL-2 cell line. The gray regions in Tables 28-39 indicatesynergism for the respective ibrutinib and JAK3 or MCL-1 inhibitorcombinations.

Table 28-29: TMD8 Cell Line

TABLE 28 ibrutinib + JAK3 Combination ibrutinib Tofacitinib (nM) (nM) CI0.0512 10000 0.324 0.256 10000 0.328 1.28 10000 0.375 6.4 10000 0.419 3210000 1.222 160 10000 4.479 800 10000 5.737 4000 10000 9.123 20000 100000.092

TABLE 29 ibrutinib + MCL-1 Combination ibrutinib MIM1 (nM) (nM) CI0.0512 3000 0.566 0.256 3000 0.558 1.28 3000 0.559 6.4 3000 0.575 323000 0.602 160 3000 0.821 800 3000 1.01 4000 3000 0.868 20000 3000 0.292

Tables 30-31: OCI-LY-10 Cell Line

TABLE 30 ibrutinib + JAK3 Combination ibrutinib Tofacitinib (nM) (nM) CI0.256 10000 9.82E+06 1.28 10000 1.91E+11 6.4 10000 4.45E+16 32 100006.82E+18 160 10000 5.95E+18 800 10000 3.55E+18 4000 10000 2.12E+18 2000010000 2.92E+18

TABLE 31 ibrutinib + MCL-1 Combination ibrutinib MIM1 (nM) (nM) CI0.0512 3000 0.019 0.256 3000 0.01 1.28 3000 0.006 6.4 3000 0.002 32 30000.003 160 3000 0.022 800 3000 0.138 4000 3000 1.304 20000 3000 3.197

TABLE 32 ibrutinib + JAK3 Combination ibrutinib Tofacitinib (nM) (nM) CI0.0512 10000 2.77E−10 0.256 10000 6.90E−09 1.28 10000 2.82E−09 6.4 100004.45E−08 32 10000 4.29E−08 160 10000 3.73E−08 800 10000 9.21E−06 400010000 2.19E−06 20000 10000 0.002

Tables 32-33: OCK-LY-3 Cell Line

TABLE 33 ibrutinib + MCL-1 Combination ibrutinib MIM1 (nM) (nM) CI0.0512 3000 4.632 0.256 3000 97.582 1.28 3000 140.961 6.4 3000 0.921 323000 2.429 160 3000 0.012 800 3000 1.771 4000 3000 2.129 20000 30004.289

Tables 34-35: HBL-1 Cell Line

TABLE 34 ibrutinib + JAK3 Combination ibrutinib Tofacitinib (nM) (nM) CI0.0512 10000 1.90E+04 0.256 10000 1.50E+04 1.28 10000 8503.943 6.4 100002.43E+04 32 10000 2.18E+04 160 10000 4.94E+04 800 10000 9.43E+04 400010000 7.17E+04 20000 10000 3.77E+06

TABLE 35 ibrutinib + MCL-1 Combination ibrutinib MIM1 (nM) (nM) CI0.0512 3000 0.231 0.256 3000 0.247 1.28 3000 0.345 6.4 3000 0.268 323000 0.333 160 3000 0.973 800 3000 0.541 4000 3000 0.676 20000 3000 0.06

Tables 36-37: U2932 Cell Line

TABLE 36 ibrutinib + JAK3 Combination ibrutinib Tofacitinib (nM) (nM) CI0.0512 10000 2485.53 0.256 10000 6308.381 1.28 10000 1.47E+05 6.4 100005417.697 32 10000 1.43E+04 160 10000 1.99E+05 800 10000 1.00E+06 400010000 5.17E+05 20000 10000 8.37E+14

TABLE 37 ibrutinib + MCL-1 Combination ibrutinib MIM1 (nM) (nM) CI0.0512 3000 4.171 0.256 3000 3.466 1.28 3000 16.388 6.4 3000 47.041 323000 92.171 160 3000 267.928 800 3000 1731.542 4000 3000 2802.807 200003000 0.189

Tables 38-39: SU-DHL-2 Cell Line

TABLE 38 ibrutinib + JAK3 Combination ibrutinib Tofacitinib (nM) (nM) CI0.0512 10000 1.827 0.256 10000 3.909 1.28 10000 6.002 6.4 10000 3.409 3210000 7.479 160 10000 3.704 800 10000 5.477 4000 10000 8.674 20000 100000.002

TABLE 39 ibrutinib + MCL-1 Combination ibrutinib MIM1 (nM) (nM) CI0.0512 3000 0.429 0.256 3000 0.402 1.28 3000 0.413 6.4 3000 0.405 323000 0.389 160 3000 0.38 800 3000 0.392 4000 3000 0.4 20000 3000 0.124

FIGS. 1A and 1B illustrate the interaction property of ibrutinib incombination with the inhibitors of MCL-1, JAK3, IDH1, and MALT1. Thecombination of ibrutinib with the MCL-1 inhibitor MIM1 was shown toexert synergistic effect in LY10 cells (red). As shown herein, thesynergistic effect (red) indicates very strong synergism (CI<0.1). Inboth TMD8 and HBL1 cells, the combination of MCL-1 inhibitor MIM1 andibrutinib sensitized these cells to ibrutinib (orange). As shown herein,the sensitize effect (orange) indicates that the ibrutinib and MIM1combination were ranged from strong synergism to slight synergism(0.1-0.9). No effects were observed for the MIM1 and ibrutinibcombination in the remaining cell lines (gray). No effect, as referredto herein, indicated that the combination did not change the sensitivityof the cells to ibrutinib. In some cases, the no effect indicated thatan antagonism was not observed.

The combination of ibrutinib with the JAK3 inhibitor tofacitinibsensitized TMD8 cells to ibrutinib (orange). No effects were observedfor the ibrutinib and tofacitinib combination for the remaining celllines (gray).

The combination of ibrutinib with the IDH1 inhibitor AGI5198 sensitizedTMD8 and LY10 cells to ibrutinib (orange). No effects were observed forthe ibrutinib and AGI5198 combination in the remaining cell lines(gray).

The combination of ibrutinib with the MALT1 inhibitor MI-2 sensitizedTMD8 and LY10 cells to ibrutinib (orange). No effects were observed forthe ibrutinib and MI-2 combination in the remaining cell lines (gray).

Example 3: Ibrutinib in Combination with Proteasome or MALT1 InhibitorsSensitizes Jeko Cells Containing a CARD11 Mutation Cell Lines

Jeko cells, a MCL cell line, and OCI-Ly3, a DLBCL cell line, were usedfor this experiment.

Stable Cell Line Generation

CARD11 mut2 contains one amino acid substitution (L244P) and mut10contains one amino acid insertion (L225LI). These two CARD11 mutantswere generated using the site-directed mutagenesis method. Wild-type(WT) or mutant (MUT) CARD11 cDNAs were inserted into the lentiviralvector pCDH-EF1. A shRNA, targeting the CARD11 3′-untranslated region toknock down the expression of the endogenous CARD11, was constructed intothe lentiviral shRNA vector pGreenPuro. Jeko cells, anibrutinib-sensitive mantle cell line, were infected with viral soupcontaining CARD11 over-expression and shRNA constructs. After infection,the cells were selected with G418 and puormycin.

DNA Sequencing Methods

Whole-exon sequencing was performed on Illumina sequencers. Exomecapture was achieved by Agilent Shureselect V4 enrichment. Reads werealigned to the hg19 reference genome using BWA and mutations identifiedusing samtools mpileup and custom filtering scripts. Substrates forSanger sequencing were PCR products produced with primers specific forCARD11 and first-strand cDNA or total DNA isolated from patient PBMC.

Combination Study

The stable cells lines were tested for the sensitivity to ibrutinib orthe combination of ibrutinib with either MALT1 or proteasome inhibitorsby 3-day CellTiter Glow assay. In summary, 10,000 cells were plated into96-well plates and treated with different concentrations of inhibitors.3 days later, CellTiter Glow reagent was added into the wells and theluminescent signals were measured.

Western Blot Analysis

The cell lysates were prepared from the cells which were treated withdifferent concentrations of inhibitors for overnight incubation.Antibodies that correlate to the proteins of interest were used fordetection.

Real-Time PCR Analysis

Total RNA was isolated from different lines and cDNA was synthesized.Primers specific to endogenous CARD11 and over-expressed CARD11s (eitherwt CARD11 or CARD11 mutants) were used to detect the expression levelsof endogenous CARD11, over-expressed CARD11 and total CARD11.

DISCUSSION

A mutation in the CARD11 gene was identified at nucleic acid residueposition 675 (FIG. 2). The mutation was a triple A insertion (FIG. 2B).Additional mutations were also observed in CARD11 (FIG. 2A). To evaluatethe functional consequence of the CARD11 mutations, Jeko cells werestably infected with either wild-type or mutant CARD11 constructs (mut2which is L244P and mut10 which is L225LI) and CARD11 shRNA which canknock-down endogenous CARD11. The modified cell lines have similarexpression levels of wild-type or mutant CARD11 which are comparable tothe endogenous CARD11 (FIG. 3). Jeko cells expressing this mutant CARD11proliferated about 40% faster than cells containing the wild-type CARD11(FIG. 4). Proliferation of Jeko cells containing the L225LI mutation(mut10) was higher in comparison to Jeko cells containing L244P (mut2)mutation or Jeko cells with the wild-type CARD11. In addition, bothmutations, L225LI and L244P, induced Jeko cells to be less sensitive toibrutinib treatment relative to Jeko cells containing wt CARD11. Thelevels of endogenous, over-expressed, and total levels of CARD11 wereexamined by real-time PCR (FIG. 5).

Proteasome inhibitors Carfilzomib and Velcade and MALT1 inhibitor MI2were tested in combination with ibrutinib to evaluate the effect of thecombination on Jeko cells containing either wild-type CARD11 or mutantCARD11 (FIG. 6). Both the proteasome inhibitors Carfilzomib and Velcadeand MALT1 inhibitor MI2 sensitized Jeko cells containing the mutantCARD11 to ibrutinib treatment. The combination of ibrutinib with eitherCarfilzomib or MI2 was further tested in OCI-Ly3 cells (a DLBCL cellline) (FIG. 7). The combination of ibrutinib with Carfilzomib sensitizedOCI-Ly3 cells (FIG. 7A), but not the combination of ibrutinib with MI2(FIG. 7B). In Jeko cells containing CARD11 L225LI (mut10) mutation, MI2was found to cause degradation of CARD11 and in some instances synergizewith ibrutinib to inhibit the NF-κB pathway (FIG. 8).

The CARD11 protein has the accession number AAI11720 and has thesequence as shown in Table 40.

TABLE 40    1mddymetlkd eedalwenve cnrhmlsryi npakltpylr qckvideqde devlnapmlp   61skinragrll dilhtkgqrg yvvfleslef yypelyklvt gkeptrrfst ivveeghegl  121thflmnevik lqqqmkakdl qrcellarlr qledekkqmt ltrvelltfq eryykmkeer  181dsyndelvkv kddnynlamr yaqlseeknm avmrsrdlql eidqlkhrln kmeeeckler  241nqslklkndi enrpkkeqvl elerenemlk tknqelqsii qagkrslpds dkaildileh  301drkealedrq elvnriynlq eearqaeelr dkyleekedl elkcstlgkd cemykhrmnt  361vmlqleever erdqafhsrd eaqtqysqcl iekdkyrkqi releekndem riemvrreac  421ivnlesklrr lskdsnnldq slprnlpvti isqdfgdasp rtngqeadds stseespeds  481kyflpyhppq rrmnlkgiql qrakspislk rtsdfqakgh eeegtdasps scgslpitns  541ftkmqpprsr ssimsitaep pgndsivrry kedaphrstv eedndsggfd aldldddshe  601rysfgpssih ssssshqseg ldaydleqvn lmfrkfsler pfrpsvtsvg hvrgpgpsvq  661httlngdslt sqltllggna rgsfvhsvkp gslaekaglr eghqlllleg cirgerqsvp  721ldtctkeeah wtiqrcsgpv tlhykvnheg yrklvkdmed glitsgdsfy irlnlnissq  781ldactmslkc ddvvhvrdtm yqdrhewlca rvdpftdhdl dmgtipsysr aqqlllvklq  841rlmhrgsree vdgthhtlra lrntlqpeea lstsdprvsp rlsrasflfg qllqfvsrse  901nkykrmnsne rvriisgspl gslarsslda tklltekqee ldpeselgkn lslipyslvr  961afycerrrpv lftptvlakt lvqrllnsgg amefticksd ivtrdeflrr qktetiiysr 1021eknpnafeci apanieavaa knkhclleag igctrdliks niypivlfir vceknikrfr 1081kllprpetee eflrvcrlke kelealpcly atvepdmwgs veellrvvkd kigeeqrkti 1141wvdedql

The CARD11 gene has the GenBank number BC111719.1 and has the sequenceas shown in Table 41.

TABLE 41 ATGGATGACTACATGGAGACGCTGAAGGATGAAGAGGACGCCTTGTGGGAGAATGTGGAGTGTAACCGGCACATGCTCAGCCGCTATATCAACCCTGCCAAGCTCACGCCCTACCTGCGTCAGTGTAAGGTCATTGATGAGCAGGATGAAGATGAAGTGCTTAATGCCCCTATGCTGCCATCCAAGATCAACCGAGCAGGCCGGCTGTTGGACATTCTACATACCAAGGGGCAAAGGGGCTATGTGGTCTTCTTGGAGAGCCTAGAATTTTATTACCCAGAACTGTACAAACTGGTGACTGGGAAAGAGCCCACTCGGAGATTCTCCACCATTGTGGTGGAGGAAGGCCACGAGGGCCTCACGCACTTCCTGATGAACGAGGTCATCAAGCTGCAGCAGCAGATGAAGGCCAAGGACCTGCAACGCTGCGAGCTGCTGGCCAGGTTGCGGCAGCTGGAGGATGAGAAGAAGCAGATGACGCTGACGCGCGTGGAGCTGCTAACCTTCCAGGAGCGGTACTACAAGATGAAGGAAGAGCGGGACAGCTACAATGACGAGCTGGTCAAGGTGAAGGACGACAACTACAACTTAGCCATGCGCTACGCACAGCTCAGTGAGGAGAAGAACATGGCGGTCATGAGGAGCCGAGACCTCCAACTCGAGATCGATCAGCTAAAGCACCGGTTGAATAAGATGGAGGAGGAATGTAAGCTGGAGAGAAATCAGTCTCTAAAACTGAAGAATGACATTGAAAATCGGCCCAAGAAGGAGCAGGTTCTGGAACTGGAGCGGGAGAATGAAATGCTGAAGACCAAAAACCAGGAGCTGCAGTCCATCATCCAGGCCGGGAAGCGCAGCCTGCCAGACTCAGACAAGGCCATCCTGGACATCTTGGAACACGACCGCAAGGAGGCCCTGGAGGACAGGCAGGAGCTGGTCAACAGGATCTACAACCTGCAGGAGGAGGCCCGCCAGGCAGAGGAGCTGCGAGACAAGTACCTGGAGGAGAAGGAGGACCTGGAGCTCAAGTGCTCGACCCTGGGAAAGGACTGTGAAATGTACAAGCACCGCATGAACACGGTCATGCTGCAGCTGGAGGAGGTGGAGCGGGAGCGGGACCAGGCCTTCCACTCCCGAGATGAAGCTCAGACACAGTACTCGCAGTGCTTAATCGAAAAGGACAAGTACAGGAAGCAGATCCGCGAGCTGGAGGAGAAGAACGATGAGATGAGGATCGAGATGGTGCGGCGGGAGGCCTGCATCGTCAACCTGGAGAGCAAGCTGCGGCGCCTCTCCAAGGACAGCAACAACCTGGACCAGAGTCTGCCCAGGAACCTGCCAGTAACCATCATCTCTCAGGACTTTGGGGATGCCAGCCCCAGGACCAATGGTCAAGAAGCTGACGATTCTTCCACCTCGGAGGAGTCACCTGAAGACAGCAAGTACTTCCTGCCCTACCATCCGCCCCAGCGCAGGATGAACCTGAAGGGCATCCAGCTGCAGAGAGCCAAATCCCCCATCAGCCTGAAGCGAACATCAGATTTTCAAGCCAAGGGGCACGAGGAAGAAGGCACGGATGCCAGCCCTAGCTCCTGCGGATCTCTGCCCATCACCAACTCCTTCACCAAGATGCAGCCCCCCCGGAGCCGCAGCAGCATCATGTCAATCACCGCCGAGCCCCCGGGAAACGACTCCATCGTCAGACGCTACAAGGAGGACGCGCCCCATCGCAGCACAGTCGAAGAAGACAATGACAGCGGCGGGTTTGACGCCTTAGATCTGGATGATGACAGTCACGAACGCTACTCCTTCGGACCCTCCTCCATCCACTCCTCCTCCTCCTCCCACCAATCCGAGGGCCTGGATGCCTACGACCTGGAGCAGGTCAACCTCATGTTCAGGAAGTTCTCTCTGGAAAGACCCTTCCGGCCTTCGGTCACCTCTGTGGGGCACGTGCGGGGCCCAGGGCCCTCGGTGCAGCACACGACGCTGAATGGCGACAGCCTCACCTCCCAGCTCACCCTGCTGGGGGGCAACGCGCGAGGGAGCTTCGTGCACTCGGTCAAGCCTGGCTCTCTGGCCGAGAAAGCCGGCCTCCGTGAGGGCCACCAGCTGCTGCTGCTAGAAGGCTGCATCCGAGGCGAGAGGCAGAGTGTCCCGTTGGACACATGCACCAAAGAGGAAGCCCACTGGACCATCCAGAGGTGCAGCGGCCCCGTCACGCTGCACTACAAGGTCAACCACGAAGGGTACCGGAAGCTGGTGAAGGACATGGAGGACGGCCTGATCACATCGGGGGACTCGTTCTACATCCGGCTGAACCTGAACATCTCCAGCCAGCTGGACGCCTGCACCATGTCCCTGAAGTGTGACGATGTTGTGCACGTCCGTGACACCATGTACCAGGACAGGCACGAGTGGCTGTGCGCGCGGGTCGACCCTTTCACAGACCATGACCTGGATATGGGCACCATACCCAGCTACAGCCGAGCCCAGCAGCTCCTCCTGGTGAAACTGCAGCGCCTGATGCACCGAGGCAGCCGGGAGGAGGTAGACGGCACCCACCACACCCTGCGGGCACTCCGGAACACCCTGCAGCCAGAAGAAGCGCTTTCAACAAGCGACCCCCGGGTCAGCCCCCGTCTCTCGCGAGCAAGCTTCCTTTTTGGCCAGCTCCTTCAGTTCGTCAGCAGGTCCGAGAACAAGTATAAGCGGATGAACAGCAACGAGCGGGTCCGCATCATCTCGGGGAGTCCGCTAGGGAGCCTGGCCCGGTCCTCGCTGGACGCCACCAAGCTCTTGACTGAGAAGCAGGAAGAGCTGGACCCTGAGAGCGAGCTGGGCAAGAACCTCAGCCTCATCCCCTACAGCCTGGTACGCGCCTTCTACTGCGAGCGCCGCCGGCCCGTGCTCTTCACACCCACCGTGCTGGCCAAGACGCTGGTGCAGAGGCTGCTCAACTCGGGAGGTGCCATGGAGTTCACCATCTGCAAGTCAGATATCGTCACAAGAGATGAGTTCCTCAGAAGGCAGAAGACGGAGACCATCATCTACTCCCGAGAGAAGAACCCCAACGCGTTCGAATGCATCGCCCCTGCCAACATTGAAGCTGTGGCCGCCAAGAACAAGCACTGCCTGCTGGAGGCTGGGATCGGCTGCACAAGAGACTTGATCAAGTCCAACATCTACCCCATCGTGCTCTTCATCCGGGTGTGTGAGAAGAACATCAAGAGGTTCAGAAAGCTGCTGCCCCGACCTGAGACGGAGGAGGAGTTCCTGCGCGTGTGCCGGCTGAAGGAGAAGGAGCTGGAGGCCCTGCCGTGCCTGTACGCCACGGTGGAACCTGACATGTGGGGCAGCGTAGAGGAGCTGCTCCGCGTTGTCAAGGACAAGATCGGCGAGGAGCAGCGCAAGACCATCTGGGTGGACGAGGACCAGCTGTGA

Example 4: Ibrutinib in Combination with MALT1 Inhibitor in a Jeko-CB17SCID Mouse Model

Jeko-CB17 SCID mice will be separated into 6 groups. Group 1 mice willbe a vehicle (i.e. control) group. Group 2 mice will be administeredwith 24 mg/kg of ibrutinib. Group 3 mice will be administered with 10mg/kg of MI2. Group 4 mice will be administered with 20 mg/kg of MI2.Group 5 will be administered a combination of ibrutinib and 10 mg/kg ofMI2. Group 6 mice will be administered a combination of ibrutinib and 20mg/kg of MI2. 10×10⁶ Jeko cells in 50% matrigel will be implanted S.C.

Example 5: Mutational Analysis of Patients with Primary Resistance toSingle-Agent Ibrutinib in Relapsed or Refractory Mantle Cell Lymphoma(MCL)

Samples were obtained from patients who participated in the MCL2001(SPARK) study, a phase 2, multicenter, single-arm study in whichpatients with MCL received ibrutinib 560 mg orally daily untilprogressive disease or unacceptable toxicity occurred. Patients who hadprogressive disease at Week 9 or earlier were considered to have primaryresistant disease. A total of 120 pateitns were examined. The patientswere further subdivided into the following categories: about 77.5% withstage IV disease, 52.5% with bulky disease, 60.0% with extranodaldisease, 41.7% with bone marrow involvement, and 9.2% with blastoidsubytpe. About 25 out of 110 patients (22.7%) were considered to haveprimary resistant disease (e.g. IRC-confirmed progressive disease atfirst disease evaluation). An additional 22 out of 110 patients (20.0%)had responses but also progressed within 12 months. These patients wereconsidered to have moderate clinical benefit. Most patients (57.3%;63/110) responded and had long durable remissions. FIG. 9 shows thepatient breakdown as progressive disease, moderate clinical benefit, orresponders.

DNA was extracted from baseline/pretreatment tumor samples (e.g. biopsyor CD19-enriched cells from peripheral blood mononuclear cells).Enriched libraries were constructed with probe sets specific for thecoding region of 97 genes possibly involved in ibrutinib response andresistance using the Ovation Target Enrichment system (NUGEN). Deepsequencing (150 bp, single-end reads) was performed with sequencesaligned to the hg19 reference genome. Possible somatic mutations wereidentified in which minor allele frequency <1% in dbSNP, >5% and <95%variant allele, and ≧10 total reads. FIG. 10 shows patient breakdownbased on clinical characteristics or on-treatment characteristics.

Sequence data were available from 23 of the 25 patients considered tohave preexisting primary resistant disease. This was based on an averageof 9 million reads. 27 genes were found with nonsynonymous variants in≧2 patients. FIG. 11 shows the set of genes observed in MCL patientsassociated with primary resistant, moderate benefit, and responders.Mutations described in CLL with acquired resistance to ibrutinib (e.g.BTK C481S, PLCγ2 R665W) were observed in the MCL patients. Genesimplicated in DLBCL pathogenesis such as MLL2 and CREBBP were observedin the MCL patients. Mutations in PIM1 and ERBB4 kinase genes wereobserved more frequent in the set of MCL patients with PD as comparedwith those patients considered as nonresistant to therapy. Several ofthe mutations detected also affected the NF-κB signaling.

In addition, patients with primary resistant disease were observed topredominantly have PIM kinase/mTOR mutations, mutations in oncogenessuch as ERBB4 and Bcl2, mutations in epigenetic modifiers such as WHSC1,MLL2 and CREBBP, and mutations in genes involved in the NF-κB (FIG. 11).

In some instances, patients with moderate clinical benefit appeared tohave more mutations in genes involved in the NF-κB pathway or BCRsignaling pathway (FIG. 11).

In some instances, patients with long durable responses appear to havefew mutations as shown in FIG. 11.

FIG. 12 illustrates analysis of the genes in primary nonresponders.

FIG. 13 illustrates a classification scheme of the genes according toNF-κB, PIM/mTOR, and epigenetic modifiers.

FIG. 14A and FIG. 14B illustrate a graphical representation of PIM1pathway (FIG. 14A) and overall survival analysis from date of diagnosiscomprising either PIM1 expression (PIM pos) or no PIM1 expression (PIMneg) (FIG. 14B). FIG. 14B is adapted from Schatz J H, et al. J Exp Med.2011:208:1799-1807.

FIG. 15 illustrates schematics of NF-κB pathways that are modulated bymutations described herein. * indicates mutations identified in Raha R.et al. Nat Med. 2014; 20:87-92. # indicates mutations identified inExample 5. FIG. 15 is adapted from Colomer D. Campo E. Cancer Cell.2014; 25:7-9.

Example 6: Analysis of PIM1, PIM2, and PIM3 Expression in ABC-DLBCL andGCB-DLBCL Cell Lines

Different ABC-DLBCL or GCB-DLBCL cell lines were tested to determine therelative endogenous gene expression of PIM1, PIM2, and PIM3.

The ABC-DLBCL cell lines included in the experiments were HBL1, TMD8,OCI-LY3, OCI-LY10, SU-DHL-2, and U-3932. The GCB-DLBCL cell linesincluded in the experiments were OCI-LY8, OCI-LY19, RCK-8, SU-DHL-1,SU-DHL-4, SU-DHL-5, SU-DHL-6, SU-DHL-8, SU-DHL-10, WSU-NHL, D8, HT, RL,and Toledo.

RT-qPCR was used to analyze gene expression.

FIGS. 16A-C illustrate a graphical representation of the relativeendogenous gene expression of PIM1, PIM2, and PIM3 in the ABC-DLBCL andGCB-DBCL cell lines tested.

Example 7: Ibrutinib Sensitivity/Resistance and PIM1 Expression in TMD8Cells and TMD8-Colony Cells

ABC-DLBCL TMD8 cells and TMD8-colony cells were used for this in vitroexperiment. TMD8-colony cells were prepared by plating TMD8 cells in0.9% methocult in a 24-well plate (1000 cells/well) and colonies of TMD8(“TMD8-colony cells”) were harvested after 7 days of incubation. Assuch, TMD8-colony cells were a subset of TMD8 cells that had increasedcolonization potential.

FIG. 17 illustrates a graphical representation of the effect ofibrutinib on relative cell growth of TMD8 and TMD8-colony cells. Asshown herein, TMD8-colony cells were more resistant to ibrutinibcompared to TMD8 cells.

FIG. 18 illustrates a graphical representation of the relative geneexpression of various genes, including PIM1. The bar graph depicts therelative gene expression as a ratio of the relative gene expression inTMD8-colony cells/relative gene expression in TMD8 cells. As shownherein, TMD8-colony cells have an increased expression of PIM1.

Example 8: Analysis of PIM1 Expression in WT andIbrutinib-Resistant-ABC-DLBCL Cells

ABC-DLBCL cell lines TMD8 and HBL1, TMD8-ibrutinib-resistant(“TMD8-resistant”), and HCL1-ibrutnib-resistant (“HBL1-resistant”) wereused for this in vitro experiment. TMD8- and HBL1-ibrutinib-resistantcells lines were generated by incubating TMD8 or HBL1 parental cellswith increasing concentrations of ibrutinib for 2 weeks. Sensitivity orresistance to ibrutinib was confirmed by the Cell-Titer-Glo® LuminescentCell Viability Assay (Promega) in accordance with manufacturer'sinstructions. TMD8 and HBL1 cell lines that were not generated to beibrutinib-resistant are also referred to as “TMD8-WT” or “HBL1-WT.”

FIG. 19A illustrates a graphical representation of the relative geneexpression of PIM1, PIM2, and PIM3 in TMD8-WT and TMD8-resistant celllines. As shown herein, the relative gene expression of PIM1, PIM2, andPIM3 is higher in TMD8-resistant cells.

FIG. 19B shows PIM1 protein expression in TMD8, HBL1, TMD8-resistant, orHBL1-resistant cells compared to protein expression of a β-actin controlin each of the foregoing. As shown herein, PIM1 protein expression isgreater in TMD8-resistant cells than TMD8-WT cells, and PIM1 proteinexpression is greater in HBL1-resistant cells than HBL1-WT cells. Assuch, PIM 1 shows differential gene expression in ibrutinib-resistantABC-DLBCL cells.

Example 9: Analysis of Synergy Between Ibrutinib and PIM Inhibitor inABC-DLBCL Cells

ABC-DLBCL cell line HBL1 was used for this in vitro experiment.HBL1-resistant cells were generated as indicated above.

The CellTiter-Glo® Luminescent Cell Viability assay was performedaccording to manufacturer's instructions. Briefly, cells were seeded at8,000-10,000 cells/well in a 96-well plate in the presence of PIMinhibitor AZD1208 or ibrutinib, either individually or in combination,for 3 days. Ibrutinib concentrations used were from 10μM in 5-folddilutions. PIM inhibitor (AZD1208) concentrations used were from 10μM in10-fold dilutions. The number of viable cells in culture was determinedby the quantification of ATP present, which was proportional to theluminal signal detected. Synergy scores and isobolograms were calculatedby the Chalice Analyzer (Horizon CombinatoRx) (FIG. 20B and FIG. 21B).As shown herein, based on the isobologram (FIG. 20B and FIG. 21B), andbased on data points and the lines falling on the left side of thediagonal line, ibrutinib and AZD1208 had synergy in both HBL1-WT andHBL-1 resistant cells.

As shown in FIGS. 20A-B and FIGS. 21A-B, ibrutinib and PIM inhibitorAZD1208 had synergy in both HBL1-WT and HBL1-resistant cells. Thesynergy scores are depicted in Table 42. A higher synergy scoreindicated better synergy.

TABLE 42 Synergy Score HBL1-WT 5.02 HBL1-resistant 7.23

FIGS. 22A-B illustrate a graphical representation of the relative cellgrowth of HBL1-WT (FIG. 23A) and HBL1-resistant (FIG. 23B) cells in thepresence of ibrutinib alone or in the presence of both ibrutinib and PIMinhibitor AZD1208. The concentration of PIM inhibitor used was 1 μM.FIG. 23C illustrates a graphical representation of the combination index(CI) for either the combination of ibrutinib and PIM inhibitor AZD1208in HBL1-WT cells, or the combination of ibrutinib and AZD1208 inHBL1-resistant cells. CI is a quantitative description of theinteraction property of the combination of two drugs. In general, thecombination is described as synergistic (CI<1), additive (CI=1), orantagonistic (CI>1). As shown herein, the combination of ibrutinib andPIM inhibitor AZD1208 showed stronger synergism in HBL1-resistant cellsthan in HBL1-WT cells.

Example 10: Analysis of Combination of Ibrutinib and PIM Inhibitor onColony Formation

ABC-DLBCL cell line HBL1-WT was used for this in vitro experiment.

HBL1-WT cells were treated with no drug, with ibrutinib alone, or withthe combination of ibrutinib and PIM inhibitor AZD1208. FIG. 23illustrates a graphical representation of the effect of no drug,ibrutinib alone, or the combination of ibrutinib and PIM inhibitorAZD1208, on colony formation of HBL1-WT cells. The combination ofibrutinib and PIM inhibitor reduced colony formation.

Example 11: Combined Drug Treatment

This animal study was completed under the Institutional Animal Care andUse Committee (IACUC)-approved protocols for animal welfare. CB17-SCIDmice (Charles Rivers Laboratories) were subcutaneously inoculated with3×10⁶ HBL1 cells in a suspension containing Matrigel (Corning). Whentumors reached approximately 100 mm³ in size, mice were randomlyassigned to one of the following treatment groups (of 9 mice each): (1)vehicle, (2) ibrutinib (24 mg/kg), (3) PIM inhibitor AZD1208 (10 mg/kg),or (4) the combination of ibrutinib (24 mg/kg) and PIM inhibitor AZD1208(10 mg/kg). Animals were treated once daily by oral gavage. Tumor volumewas measured twice a week and calculated as tumorvolume=(length×width²)×0.4. Tumor size over 18 days is shown for eachtreatment group in FIGS. 24B-25E, with average values shown in FIG. 25A.As shown herein, the combination of ibrutinib and PIM inhibitor enhancedthe growth suppression effect of ibrutinib on HBL1 tumors/xenografts.

Example 12: Mutational Analysis of Patients with ABC-DLBCL

Samples were obtained from DLBCL patients who participated in clinicaltrail NCT00849654 or clinical trial NCT01325701. NCT00849654 was a phase1 dose-escalation study of ibrutinib in recurrent B-cell lymphoma, andNCT01325701 was a multi-center phase 2 study of ibrutinib in patientswith relapsed and refractory or de novo DLBCL. A total of 48 DLBCLpatients were examined for PIM1 mutations. Targeted deep sequencing wasused to determine the impact of baseline mutations in PIM1 on clinicalresponse to ibrutinib. H&E-stained slides of each patient were reviewedto ensure sufficient nucleated cellularity and tumor content. DNA andRNA were extracted from unstained sections of FFPE DLBCL tumor biopsies.Sequencing was performed using FoundationOne Heme™ panel(FoundationOne®) following Next-Generation Sequencing (NGS)-basedprotocol (Illumina) in accordance with the manufacter's instructions.The heme panel validated the NGS-based protocol to interrogate completecoding DNA sequences of 405 genes as well as selected introns of 31genes involved in rearrangements. Sequence data were processed andanalyzed to check for base substitutions, insertions, deletions,copy-number alterations, and select gene fusions. Mutation impactindices of 317 genes were calculated and plotted for overall genemutation pattern recognition. Chi-square association tests wereperformed on cases where sufficient sample sizes were available todetermine statistical significance of mutation impact. Gene expressionprofiling (GEP) and Hans' Immunohistochemistry algorithm were used toinvestigate DLBCL subtype classifications. Omnisoft Corporation's ArrayStudio software was used to build a linear discriminant analysis (LDA)model/classifier and neural networks (NNs) with 5-fold cross-validationprocedure for model selection. LDA was selected for final GEPclassification.

FIG. 25A shows PIM1 mutations observed in 6 patients, 5 ABC-DLBCLpatients, and 1 GCB-DLBCL patient. PIM1 P81S, PIM1 S97N, and PIM1 L2Vmutations were found in ABC-DLBCL patients with progressive disease (PD)following ibrutinib treatment. As shown herein, the foregoing PIM1mutations can be indicative of ibrutinib resistance. Additionally, PIM1mutations appeared more frequently in patients diagnosed with ABC-DLBCLcompared to patients diagnosed with GCB-DLBCL. 5 out of 6 patients withPIM mutations were ABC-DLBCL patients. Of these 5 patients, 4 exhibiteda poor clinical response to ibrutinib (i.e., 80% of ABC-DLBCL patientswith PIM1 mutations have progressive disease (PD), compared to only 13out of 26 (i.e., 50%) of ABC-DLBCL patients without PIM1 mutations havePD. See Tables 43-46 below for polypeptide sequence of PIM1-WT and PIM1mutants.

TABLE 43 PIM1-WT (SEQ. ID NO.: 1)MLLSKINSLAHLRAAPCNDLHATKLAPGKEKEPLESQYQVGPLLGSGGFGSVYSGIRVSDNLPVAIKHVEKDRISDWGELPNGTRVPMEVVLLKKVSSGFSGVIRLLDWFERPDSFVLILERPEPVQDLFDFITERGALQEELARSFFWQVLEAVRHCHNCGVLHRDIKDENILIDLNRGELKLIDFGSGALLKDTVYTDFDGTRVYSPPEWIRYHRYHGRSAAVWSLGILLYDMVCGDIPFEHDEEIIRGQVFFRQRVSSECQHLIRWCLALRPSDRPTFEEIQNHPWMQDVLLPQETA EIHLHSLSPGPSK

TABLE 44 PIM1 L2V (SEQ. ID NO. 2) M VLSKINSLAHLRAAPCNDLHATKLAPGKEKEPLESQYQVGPLLGSGGFGSVYSGIRVSDNLPVAIKHVEKDRISDWGELPNGTRVPMEVVLLKKVSSGFSGVIRLLDWFERPDSFVLILERPEPVQDLFDFITERGALQEELARSFFWQVLEAVRHCHNCGVLHRDIKDENILIDLNRGELKLIDFGSGALLKDTVYTDFDGTRVYSPPEWIRYHRYHGRSAAVWSLGILLYDMVCGDIPFEHDEEIIRGQVFFRQRVSSECQHLIRWCLALRPSDRPTFEEIQNHPWMQDVLLPQETA EIHLHSLSPGPSK

TABLE 45 PIM1 S97N (SEQ. ID NO. 3)MLLSKINSLAHLRAAPCNDLHATKLAPGKEKEPLESQYQVGPLLGSGGFGSVYSGIRVSDNLPVAIKHVEKDRISDWGELPNGTRVPMEVVLLKKV N SGFSGVIRLLDWFERPDSFVLILERPEPVQDLFDFITERGALQEELARSFFWQVLEAVRHCHNCGVLHRDIKDENILIDLNRGELKLIDFGSGALLKDTVYTDFDGTRVYSPPEWIRYHRYHGRSAAVWSLGILLYDMVCGDIPFEHDEEIIRGQVFFRQRVSSECQHLIRWCLALRPSDRPTFEEIQNHPWMQDVLLPQETA EIHLHSLSPGPSK

TABLE 46 PIM1 P81S (SEQ. ID NO. 4)MLLSKINSLAHLRAAPCNDLHATKLAPGKEKEPLESQYQVGPLLGSGGFGSVYSGIRVSDNLPVAIKHVEKDRISDWGEL S NGTRVPMEVVLLKKVSSGFSGVIRLLDWFERPDSFVLILERPEPVQDLFDFITERGALQEELARSFFWQVLEAVRHCHNCGVLHRDIKDENILIDLNRGELKLIDFGSGALLKDTVYTDFDGTRVYSPPEWIRYHRYHGRSAAVWSLGILLYDMVCGDIPFEHDEEIIRGQVFFRQRVSSECQHLIRWCLALRPSDRPTFEEIQNHPWMQDVLLPQETAEIHLHS LSPGPSK

FIG. 25B illustrates a schematic of the kinase domain of PIM1. It alsoincludes a list of PIM1 mutations that were identified in DLBCL patientswho participated in the clinical trials indicated above.

Example 13. In Vitro Analysis of Functional Consequences of PIM1Mutations

PIM1 mutations were generated using the site-directed mutagenesismethod. Wild-type (WT) or mutant (MUT) PIM1 cDNAs were inserted intolentiviral vector pCDH (FIG. 26). 293T cells were transfected with pCDHcontructs. Two days after transfection, the cells were used for theprotein stability assay. These cell lines also referred to herein as“modified cell lines” or “modified cells.”

To evaluate the functional consequence of the PIM1 mutations, thecycloheximide cell assay was used to evaluate protein stability in themodified cell lines. Briefly, comparison of protein stability ineukaryotic cells can be achieved by cycloheximide, which is an inhibitorof protein biosynthesis. After cycloheximide treatment, proteinexpression was evaluated by Western Blot (FIGS. 27A-E). Antibodies thatcorrelate to the protein of interest were chosen for detection.Cycloheximide treatment is identified as “CHX” in FIGS. 27A-E. As shownin FIG. 27A, in modified cells expressing PIM1-WT, the expression ofPIM1 WT was reduced with cycloheximide treatment. However, in modifiedcells expressing mutant PIM1, the expression of mutant PIM1 was notreduced, or stayed relatively the same, with cycloheximide treatment(FIGS. 27B-E). As shown herein, PIM1 mutations confer protein stability.

Example 14: In Vitro Analysis of PIM1 Mutations in ABC-DLBCL CellsTreated with Ibrutinib

PIM1 mutations were generated using the site-directed mutagenesis methodas described above. Wild-type (WT) or mutant (MUT) PIM1 cDNAs wereinserted into lentiviral vector pCDH (FIG. 26). TMD8 cells were infectedwith pCDH contructs. After infection, the cells were selected withpuormycin. These cell lines also referred to herein as “modified celllines” or “modified TMD8 cells.”

In this manner, modified TMD8 cells expressing PIM1-WT, PIM1 L2V, PIM1P81S, PIM1 S97N were generated. The modified TMD8 cells were treatedwith ibrutinib, and cell growth was measured. As shown in FIGS. 28 andTable 46, TMD8 cells expressing mutant PIM1 proteins were more resistantto ibrutinib treatment than TMD8 cells expressing PIM1-WT.

TABLE 46 PIM1 EC50 (nM) WT 76 L2V >1000 P81S >1000 S97N >1000

To evaluate whether the relative cell growth amongst the differentmodified TMD8 cell lines was indeed due to resistance to ibrutinib ordue to growth rate and viability conferred by the different proteinsirrespective of ibrutinib treatment, cell number and cell viability ofeach of the four groups of modified TMD8 cells was measured (FIGS.30A-B) without ibrutinib treatment. No significant difference was seenin cell growth and variability amongst the four modified TMD8 cell lines(FIGS. 30A-B) in the absence of ibrutinib.

A clonogenic cell survival assay was performed to evaluate whether anydifferences in the ability to proliferate indefinitely exists amongstthe different modified TMD8 cell lines (FIGS. 30A-F). Modified TMD8cells expressing mutant PIM1 showed increased clonogenicity in theabsence of ibrutinib (FIG. 30A). Addition of ibrutinib to each modifiedTMD8 cell line reduced clonogenicity (FIG. 30B); however, clonogenicitywas reduced to a greater extent in modified TMD8 cells expressingPIM1-WT. FIGS. 30C-F illustrate microscope views (100× magnification) ofmodified TMD8 cells expressing the following: PIM1 WT (FIG. 30C); PIM1L2V (FIG. 30D); PIM1 P81S (FIG. 30E); and PIM1 S97N (FIG. 30F).

Based on the foregoing, PIM1 protein levels may be increased due to theincreased half-life of mutant PIM1 proteins, and/or may be increased dueto gene up-regulation of PIM1-WT.

Additionally, analysis of the effect of PIM1 mutations on downstreamsignaling in DLBCL cell lines can be conducted. Phosphorylation levelsof PIM1 targets, cytokine/chemokin secretion from the cells and geneexpression changes through microarray analysis can be studied. SincePIM1 phosphorylates NF-kB P65, gene expression of NF-kB can be studied.

The examples and embodiments described herein are for illustrativepurposes only and various modifications or changes suggested to personsskilled in the art are to be included within the spirit and purview ofthis application and scope of the appended claims.

1. A method of treating a B-cell malignancy in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of a combination comprising a BTK inhibitor and ananticancer agent, wherein the anticancer agent inhibits MALT1, MCL-1, orIDH1.
 2. The method of claim 1, wherein the B-cell malignancy is a BTKinhibitor-resistant B cell malignancy.
 3. (canceled)
 4. The method ofclaim 1, wherein the combination sensitizes a B-cell malignancy to theBTK inhibitor.
 5. The method of claim 1, wherein the anticancer agentinhibits MALT1.
 6. The method of claim 5, wherein the anticancer agentthat inhibits MALT1 comprises MI-2, mepazine, thioridazine, andpromazine.
 7. The method of claim 1, wherein the anticancer agentinhibits MCL-1.
 8. The method of claim 7, wherein the anticancer agentthat inhibits MCL-1 comprises BI97C10, BI112D1, gossypol, obatoclax,MG-132, MIM1, sabutoclax, and TW-37.
 9. The method of claim 1, whereinthe anticancer agent inhibits IDH1.
 10. The method of claim 9, whereinthe anticancer agent that inhibits IDH1 comprises AGI-5198, AG-120,IDH-C227, and ML309.
 11. The method of claim 1, wherein the BTKinhibitor is ibrutinib.
 12. The method claim 1, wherein the B-cellmalignancy is acute lymphoblastic leukemia (ALL), acute myelogenousleukemia (AML), chronic myelogenous leukemia (CIVIL), acute monocyticleukemia (AMoL), chronic lymphocytic leukemia (CLL), small lymphocyticlymphoma (SLL), high-risk small lymphocytic lymphoma (SLL), follicularlymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle celllymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma,extranodal marginal zone B cell lymphoma, nodal marginal zone B celllymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma,primary mediastinal B-cell lymphoma (PMBL), immunoblastic large celllymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocyticleukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma,plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B celllymphoma, intravascular large B cell lymphoma, primary effusionlymphoma, or lymphomatoid granulomatosis.
 13. The method of claim 12,wherein the B-cell malignancy is diffuse large B-cell lymphoma (DLBCL)or activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL). 14.(canceled)
 15. The method of claim 1, wherein the method furthercomprises administering a third therapeutic agent.
 16. The method ofclaim 15, wherein the third therapeutic agent is selected from among achemotherapeutic agent or radiation therapeutic agent.
 17. (canceled)18. A pharmaceutical combination comprising: a) a BTK inhibitor; b) ananticancer agent, wherein the anticancer agent inhibits MALT1, MCL-1,IDH1, or proteasome; and c) a pharmaceutically-acceptable excipient. 19.A method of treating a B-cell malignancy in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of a combination comprising a BTK inhibitor and a PIM inhibitor.20. The method of claim 19, wherein the combination provides asynergistic effect compared to administration of the BTK inhibitor orthe PIM inhibitor alone.
 21. The method of claim 20, wherein thecombination sensitizes the B-cell malignancy to the BTK inhibitor. 22.The method of claim 19, wherein the BTK inhibitor is ibrutinib.
 23. Themethod of claim 19, wherein the PIM inhibitor comprises mitoxantrone,SGI-1776, AZD1208, AZD1897, LGH447, JP_11646, Pim1 inhibitor 2,SKI-O-068, CX-6258, AR460770, AR00459339 (Array Biopharma Inc.),miR-33a, Pim-1 inhibitory p27 (Kip1) peptide, LY333′531, K00135,quercetagein (3,3′,4′,5,6,7-hydroxyflavone), or LY294002.
 24. (canceled)